Antibodies, Kit and Method for Detecting Amyloid Beta Oligomers

ABSTRACT

This invention is a selective Aβ oligomer kit and immunoassay method capable of reliably and sensitively detecting Aβ oligomers in a biological sample of a patient. In one embodiment the inventive assay uses a pair of anti-Aβ oligomer antibodies, as capture and detection antibodies, to detect and quantify Aβ oligomers. The method can be used to differentiate Alzheimer&#39;s disease (AD) patients from non-AD patients and/or to stratify AD patients according to the severity of their disease.

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/507,332 filed Jul. 13, 2011, and is a continuation-in partapplication of PCT/US2011/043866, filed Jul. 13, 2011, which claims thebenefit of priority from U.S. Provisional Patent Application Ser. No.61/364,210, filed Jul. 14, 2010, the contents of which are incorporatedherein by reference in their entireties.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a devastating neurodegenerative diseasecharacterized by amyloid β (Aβ) plaque accumulation in brain regionsinvolved in learning and memory. While these large insoluble plaqueswere once thought to cause AD, evidence now indicates that smalldiffusible oligomers of Aβ may be responsible. Amyloid-deriveddiffusible ligands (ADDLs) are a species of Aβ oligomers that can begenerated in vitro with properties similar to endogenous Aβ oligomers(U.S. Pat. No. 6,218,506; Klein, et al. (2004) Neurobiol. Aging25:569-580; Lambert, et al. (1998) Proc. Natl. Acad. Sci. USA95:6448-6453). Aβ oligomers are present in the brain of AD patients,they bind neurons, and they induce deficits in neuronal morphology andmemory. Studies with antibodies that bind Aβ oligomers have shownimprovement in both neuronal morphology and memory.

Assays to measure Aβ monomers are known. For example, a sandwich ELISAcomposed of N-terminus (Aβ1) end-specific antibody (clone 82E1) andC-termini end-specific antibodies for Aβ1-40 (clone 1A10) and Aβ1-42(clone 1C3) was developed to detect full-length Aβ1-40 and Aβ1-42 with asensitivity in the sub-single digit fmol/ml (equivalent to single digitpg/ml) range with no cross-reactivity to APP (Horiskoshi, et al. (2004)Biochem. Biophys. Res. Commun. 319:733-737 and US Patent Publication No.2011/0008339). Additional assays have used used the activity of β- andγ-secretase enzymes on the amyloid precursor protein (APP) to detectmonomers; however, few assays have been reported that specifically andreliably detect Aβ oligomers in a human fluid sample, such ascerebrospinal fluid (CSF), in both normal control and in AD(Georganopoulou, et al. (2005) Proc. Natl. Acad. Sci. USA 102:2273-2276;Fukumoto, et al. (2010) FASEB J. 24:2716-2726; Gao, et al. (2010) PLoSOne 5(12):e15725). Reported Aβ oligomer assays have employed a number ofapproaches, including ADDL-specific antibodies coupled with abio-barcode PCR amplification platform (Georganopoulou, et al. (2005)supra), overlapping epitope ELISAs (Gandy, et al. (2010) Ann. Neurol.68:220-230; Xia, et al. (2009) Arch. Neurol. 66:190-199), also pairedfirst with size exclusion chromatography (Fukomoto, et al. (2010)supra), and amyloid-affinity matrices methods (Gao, et al. (2010) supra;Tanghe, et al. (2010) Int. J. Alz. Dis. 2010:417314), followed byoligomer dissociation and measurement with antibodies to Aβ monomers.

Aβ oligomers have also been detected from either CSF or brain using gelelectrophoresis followed by western blot analysis (Klyubin, et al.(2008) J. Neurosci. 28:4231-4237; Hillen, et al. (2010) J. Neurosci.30:10369-10379), or subsequent to size exclusion chromatography(Shankar, et al. (2011) Methods Mol. Biol. 670:33-44), relying on themolecular weight of oligomers that are maintained after theelectrophoretic procedure. However, electrophoretic and blottingtechniques do not provide the sensitivity required to see these speciesin normal control CSF (Klyubin, et al. (2008) supra), which exhibit a1000-fold range of Aβ oligomer concentrations (Georganopoulou, et al.(2005) supra). Aβ oligomer species represent a wide range of molecularweights and, as such, assignment of a precise molarity is problematic.While a lower limit of detection at 100 aM has been shown(Georganopoulou, et al. (2005) supra), most reported methods(Georganopoulou, et al. (2005) supra; Gao, et al. (2010) supra;Fukumoto, et al. (2010) supra; Gandy, et al. (2010) supra) do not assessselectivity between signals from Aβ oligomers as compared to Aβmonomers, so the concentrations noted should be viewed with caution. Oneassay (Xia, et al. (2009) Arch. Neural. 66:190-199), marketed byImmunobiological Laboratories, Inc. (Minneapolis, Minn.) claims 320-foldselectivity for Aβ1-16 dimers as compared to Aβ40 monomer, but lacks theselectivity needed to avoid cross-reactivity with Aβ monomer in the CSF.As Aβ oligomers in the CSF are hypothesized to be present at fM levelsand CSF Aβ monomers are present between 1.5-2 nM, an assay thatselectively measures Aβ oligomers in a CSF sample must have exceptionalselectivity for Aβ oligomers over monomers.

Aβ oligomers have also been used as a target for therapeutic monoclonalantibodies to treat AD (see, for example, U.S. Pat. Nos. 7,811,563,7,780,963, and 7,731,962). It is believed that these antibodies accessthe central nervous system (CNS) and clear the toxic ADDL species fromthe brain, through 1) catalytic turnover by Fc-mediated activation ofmicroglia, 2) clearance of antibody/ADDL complexes into thecerebro-vasculature, or 3) enzymatic digestion of the ADDLs followingantibody binding and improved access of degradative enzymes, such asneprilysin, insulin-degrading enzyme, plasmin, endothelin-convertingenzymes (ECE-1 and -2), matrix metalloproteinases (MMP-2, -3 and -9),and angiotensin-converting enzyme (ACE). Thus, a goal of a selective Aβoligomer assay is to measure the pharmacodynamic (PD) change in CNS Aβoligomers following treatment with an anti-oligomer antibody or othertreatment that alters Aβ monomer/oligomer formation or clearance.Additionally, an assay that would specifically enable the detection ofAβ oligomers bound to an anti-Aβ oligomer antibody, i.e., a targetengagement (TE) assay, would be invaluable for the assessment of thetherapeutic antibody following treatment.

SUMMARY OF THE INVENTION

The present invention is directed to a selective Aβ oligomer kit andmethod capable of reliably and sensitively detecting Aβ oligomers in abiological sample, e.g., a fluid sample, of a subject. The inventive kitand method use a pair of highly selective anti-Aβ oligomer antibodies todetect and quantify A3 oligomers in a biological sample. In particular,the kit and method of the invention employ a capture antibody that (i)recognizes an N-terminal linear epitope of amyloid beta 1-42 peptide,e.g., an epitope within residues 1-20 of amyloid beta 1-42, (ii)recognizes a conformational epitope of amyloid beta 1-42 oligomers,(iii) has a higher affinity for amyloid beta 1-42 oligomers than foramyloid beta 1-42 monomer, amyloid beta 1-40 monomer, plaques andamyloid beta fibrils, (iv) exhibits less than a 10-fold decrease in EC₅₀when stored at 40° C. for 1 month; and a detection antibody thatrecognizes an N-terminal linear epitope of amyloid beta 1-42 peptide,e.g., an epitope within residues 1-20 of amyloid beta 1-42. In someembodiments, the affinity of the capture antibody for amyloid beta 1-42oligomers compared to amyloid beta 1-40 monomers in a competitivebinding assay is at least 500:1. In other embodiments, the affinity ofthe capture antibody for amyloid beta 1-42 oligomers compared to amyloidbeta 1-42 monomers in a sandwich ELISA assay is at least 500:1 or atleast 1000:1. In still other embodiments, the capture antibody is avariant of antibody h3B3 or a variant of antibody 19.3. In certainembodiments, the detection antibody is 6E10, BAM-10, W0-2, 26D6, 2A10,2B4, 4C2, 4E2, 2H4, 20C2, 2D6, 5F10, 1F4, 1F6, 2E12, 3B3 or 82E1, andcan optionally include a label. Using the kit and method, optionally incombination with a means for concentrating an antibody-antigen complex,the level of detection of amyloid beta 1-42 oligomers is less than 5pg/mL or less than 3 pg/mL. Isolated antibodies, or antibody fragments,of use in the kit and method of the invention are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation showing the selectivity of theanti-ADDL antibodies binding to the ADDL species of Aβ oligomers (middlebar of each set), as compared to Aβ monomer or Aβ fibril. Shown is ELISAbinding of a panel of humanized (h3B3) and affinity matured anti-ADDL(14.2, 7.2, 11.4, 9.2, 13.1, 17.1, and 19.3) antibodies and threecomparator antibodies (Comp 1, 2, and 3) to monomeric Aβ, ADDLs andfibrillar Aβ. Comparative antibody 2 is known to be non-selectiveantibody for ADDLs. The background of this assay was determined byremoving the capture antibody from the ELISA (no mAb). Error barsrepresent standard error of the mean.

FIG. 2 is a graphic representation of the ELISA binding of anti-ADDLantibody 19.3 and antibody 3B3 to ADDLs or monomer Aβ (Aβ1-40) evaluatedwith an 11 point titration curve.

FIG. 3 is a graphic representation of the ability of anti-ADDL antibody19.3 and 3B3 to block ADDL binding to primary hippocampal neuronal cellsafter pre-incubation with increasing concentration of the antibody. Theability of anti-ADDL antibody 19.3 to block ADDL binding to neurons wasattenuated after heat denaturing of the antibody. Error bars representstandard error of the mean.

FIG. 4 is a graphic representation of the binding and dissociation ofanti-ADDL antibodies to immobilized human FcRn when assessed withBIACORE (GE Healthcare, Piscataway, N.J.). The adjusted sensorgram showsinitial binding at pH 6.0 and then the dissociation of antibodies at pH7.3 from 180 seconds. A report point (Stability) was inserted at 5seconds after the end of pH 6.0 binding and the “% bound” was calculatedas RU_(stability)/RU_(Binding) (%).

FIG. 5A shows a one-sided ELISA with plates coated with either Aβoligomer (triangles) or Aβ monomer (squares), demonstrating the relativeaffinities and maximum binding characteristics of the humanized antibody19.3.

FIG. 5B shows a competitive ELISA and the relative affinities of 19.3for Aβ oligomers (triangles) and Aβ monomer (squares) coated on an ELISAplate in the presence of the competing species in solution.

FIGS. 6A-6C are graphic representations of the ELISA binding to ADDLs ofthe anti-ADDL antibody 19.3 (designated as WT in FIG. 6A) and two19.3-derived anti-ADDL antibodies (FIGS. 6B and 6C) after incubation upto one month at varying temperatures to evaluate antibody stability. The19.3-derived anti-ADDL antibodies were composed of a single amino-acidsubstitution of Asn33 within light chain CDR1 to either Ser33 (N33S;FIG. 6B) or Thr33 (N33T; FIG. 6C). Substitution of Asn33 with either S33or T33 resulted in improved antibody stability versus the parental 19.3antibody.

FIGS. 7A-7C are graphic representations showing the sensitivity of threepairs of antibodies in a sandwich ELISA format using chemiluminesence(ENVISION Multilabel Reader, Perkin Elmer, Waltham, Mass.), as thedetection method and their relative affinities for Aβ oligomers. FIG. 7Ashows the anti-Aβ oligomer antibody 19.3 as the capture antibody and82E1 as the detection antibody over a range of Aβ oligomerconcentrations. FIGS. 7B and 7C depict 6E10 and 19.3, respectively, asboth the capture and detection antibodies. The 19.3×82E1 sandwich ELISApair (FIG. 7A) was significantly more sensitive in detecting A3oligomers as compared to other pairs (FIGS. 7B and 7C).

FIG. 8 is a graphic representation of the sensitivity and selectivityfor the detection of Aβ oligomers (squares) as compared to Aβ monomer(triangles) using the anti-Aβ oligomer antibodies 19.3 and 82E1 asmeasured using a paramagnetic microparticle detector, such as the ERENNAdigital detector (SINGULEX, Almeda, Calif.). Use of the paramagneticmicroparticle detector significantly improved the sensitivity to detectAβ oligomers with the 19.3/82E1 antibody pair.

FIGS. 9A and 9B are graphic representations of the Aβ oligomer sandwichELISA, i.e., the Pharmacodynamic (PD) Assay, and the Aβoligomer/antibody sandwich ELISA, i.e., the Target Engagement Assay,respectively.

FIGS. 10A and 10B are graphic representations of the levels of Aβoligomers detected in human cerebrospinal fluid (CSF) samples. FIG. 10Ashows that the Aβ oligomers levels were four-fold higher in AD patientsas compared to age matched control, i.e., non-AD, patients in a blindedevaluation using the method herein. The differences were statisticallysignificant to p≦0.0004 as determined using a two-way t-test and MannWhitney analysis of ranks, assuming the population was non-Gaussian.FIG. 10B shows that the Aβ oligomer levels were eight-fold higher in ADpatients as compared to young control, i.e., non-AD, patients in ablinded evaluation using the method herein. The differences were alsostatistically significant between these groups using the samestatistical method as in FIG. 10A to a p-value ≦0.0021.

FIGS. 11A and 11B are graphic representations of Aβ monomer levels inthe CSF of either clinically confirmed AD or young control, i.e.,non-AD, patients, with a corresponding decrease in the levels of Aβ1-42monomer and unchanged levels of Aβ1-40 monomer in the AD samples. Thisis representative of the general pattern observed for AD patients andconfirmed the disease state of the samples evaluated in FIG. 10B. FIG.11A shows the reduced levels of Aβ1-42 monomer in the AD CSF samples.The differences were statistically significant to p≦0.002 as determinedusing a two-way t-test and Mann Whitney analysis of ranks, assuming thepopulation was non-Gaussian. FIG. 11B shows the unchanged levels betweenthe two groups of Aβ1-40 monomer.

FIG. 12 is a graphic representation of the correlation betweenMini-Mental State Exam (MMSE) scores, as a measure of cognitiveperformance, and levels of Aβ oligomer measured using the assaydescribed herein. All patients depicted in FIG. 10B were included inthis correlation. The correlation at −0.7445 pg/mL of Aβ oligomers wassignificant with p≦0.0001.

FIG. 13 is a graphical representation of the PK of anti-ADDL antibody19.3 assessed in primate (three male rhesus monkeys) cerebrospinal fluid(CSF) using a cisterna magna ported rhesus model followingadministration of a bolus IV dose of 20 mg/kg. At about 24 hours postdose, antibody 19.3 was present in the CSF at 100 ng/mL.

FIGS. 14A and 14B are graphical representations of the target engagementassay. FIG. 14A is a representation of anti-Aβ oligomer antibody 19.3/A3oligomer complexes formed ex vivo with spiking into human CSF (circle)or casein buffer (triangle). FIG. 14B is a representation of anti-Aβoligomer antibody 19.3/Aβ oligomer complexes formed ex vivo with spikinginto human CSF (circle) or Casein buffer (triangle). Differentialsensitivity was observed in the detection of 19.3/Aβ oligomer complexesin an anti-human kappa chain (capture)×82E1 (detection) targetengagement ELISA. The anti-kappa capture antibody poorly differentiatedthe anti-Aβ oligomer antibody 19.3 from the endogenous antibody speciesin human CSF.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a kit and method for reliably and sensitivelydetecting Aβ oligomers in a biological sample, such as the CSF of apatient for use as both a pharmacodynamic and target engagement measureof Aβ oligomers. The kit and method of the invention can differentiateAD from non-AD patients and stratify AD disease state based on elevatedlevels of Aβ oligomers in, for example the CNS of the AD patients,similar to uses previously reported for a tau/Abeta42 CSF ratio (DeMeyer, et al. (2010) Arch. Neurol. 67:949-56). Moreover, an Aβ oligomerassay, detecting the most neurotoxic species, may correlate better andbe a more dynamic measure of changes in cognitive performance, ascompared to the poor correlation observed for levels of Aβ monomer. Ithas now been demonstrated that a peripherally administered anti-Aβoligomer antibody can penetrate the blood-brain-barrier and bind Aβoligomers and, when used in combination with the method herein, canprovide a surrogate end-point assay for the assessment of ADtherapeutics.

For the purposes of this invention, the term “Aβ oligomers” refers tomultimer species of Aβ monomer that result from self-association ofmonomeric species. Aβ oligomers are predominantly multimers of Aβ1-42,although Aβ oligomers of Aβ1-40 have been reported. Aβ oligomers mayinclude a dynamic range of dimers, trimers, tetramers and higher-orderspecies following aggregation of synthetic Aβ monomers in vitro orfollowing isolation/extraction of Aβ species from human brain or bodyfluids. ADDLs are one species of Aβ oligomers.

The term “ADDLs” or “amyloid β-derived diffusible ligands” or “amyloidβ-derived dementing ligands” as used herein refers to a neurotoxic,soluble, globular, non-fibrillar oligomeric structure containing two ormore Aβ protein monomers. Higher order oligomeric structures can beobtained not only from Aβ1-42, but also from any Aβ protein capable ofstably forming the soluble non-fibrillar Aβ oligomeric structures, suchas Aβ1-43 or Aβ1-40. See U.S. Pat. No. 6,218,506 and WO 01/10900.

The term “Aβ fibrils” or “fibrils” or “fibrillar amyloid” as used hereinrefers to insoluble species of Aβ that are detected in human andtransgenic mouse brain tissue because of their birefringence with dyessuch as thioflavin S. Aβ species that form fiber-like structurescomprised of Aβ monomers include β-pleated sheets. These species arebelieved to be immediate precursors to the extracellular amyloid plaquestructures found in AD brain.

The term “Aβ1-40 monomer” or “Aβ1-42 monomer” as used herein refers tothe direct product of the enzymatic cleavage, i.e., aspartic proteaseactivity, by β-secretase and γ-secretase on the amyloid proteinprecursor (APP) in a cell-free or cellular environment. Cleavage of APPby β-secretase generates the Aβ species beginning at Asp 1 (numbering asto Aβ peptide sequence after cleavage), while γ-secretase liberate theC-terminus of Aβ, predominantly either at residues 40 or 42.

A highly sensitive assay has now been developed to detect and measurethe levels of Aβ oligomers in a biological sample, e.g., a fluid sample,preferably CSF. The kit and method of the invention use two anti-Aβoligomer-selective antibodies as capture and detection antibodies in acompetitive binding assay, such as a sandwich ELISA. The term “captureantibody” or “Aβ oligomer capture antibody” or “anti-human IgG2 captureantibody” as used herein refers to an antibody that is used as thecapture antibody in the assays herein. The capture antibody as usedherein binds to an Aβ oligomer or Aβ oligomer/antibody complex that arebeing measured and/or detected in a sample.

According to the kit and method, the capture antibody is characterizedas recognizing an N-terminal linear epitope of amyloid beta 1-42peptide, having a higher affinity for amyloid beta 1-42 oligomers thanfor amyloid beta 1-42 monomer, amyloid beta 1-40 monomer, plaques andamyloid beta fibrils, and exhibiting exhibits less than a 10-folddecrease in EC₅₀ when stored at 40° C. for month; and the detectionantibody is characterized as recognizing an N-terminal linear epitope ofamyloid beta 1-42 peptide.

As is known in the art, a linear epitope is an epitope, wherein an aminoacid primary sequence includes the epitope recognized. A linear epitopetypically includes at least 3, and more usually, at least 5, forexample, about 8 to about 10 amino acids in a unique sequence. Inparticular embodiments of this invention, the capture and detectionantibody both recognize a linear epitope at the N-terminus of the Aβ1-42peptide and this linear epitope may be the same or different. In certainembodiments, the, or each, linear epitope is located within residues1-20 of Aβ1-42 peptide, or in the N-terminal 10, 11, 12, 15 or 20 aminoacid residues of amyloid β1-42. In particular embodiments, an antibodyof the invention specifically binds to a linear epitope within residues1-5, 1-8, 1-10, 1-20, 3-8, or 3-10 of amyloid β1-42 and this linearepitope may be the same or different for each of the capture anddetection antibody.

The linear epitope of an antibody can be readily mapped by generating aset of overlapping, five-ten amino acid peptides of Aβ1-42, anddetermining binding of the antibody the set of peptides in a competitivebinding assay, such as an ELISA assay. Using such an assay the corelinear epitope of various commercial antibodies have been determined.Based upon the analysis presented in U.S. Pat. No. 7,780,963 andHorikoshi, et al. (2004) supra, the linear epitopes of antibodies of usein this invention are presented in Table 1.

TABLE 1 SEQ Core Epitope Sequence within Aβ1-42  ID NO: Antibody EpitopeDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA 7 6E10  5-11 RHDSGYE 8 BAM-103-8 EFRHDS 9 4G8 xx-21 EVHHQKLVFFA 10 WO-2 3-8 EFRHDS 9 26D6 3-8 EFRHDS9 2A10^(a) 3-8 EFRHDS 9 2B4^(b) 3-8 EFRHDS 9 4C2^(a) 3-8 EFRHDS 94E2^(a) 3-8 EFRHDS 9 2H4^(c) 1-8 DAEFRHDS 11 20C2^(a) 3-8 EFRHDS 92D6^(a) 3-8 EFRHDS 9 5F10^(c) 3-8 EFRHDS 9 1F4^(a) * 1F6^(a) * 2E12^(a) 3-10 EFRHDSGY 12 3B3^(a) * 82E1 1-5 DAEFR 13 Core epitope position iswith respect to Aβ1-42. ^(a)IgGl, ^(b)IgG2b, ^(c)IgG2a. *Epitopes wereestimated to be located at the N-terminus of Aβ1-42, as they could bindto Aβ1-20 peptide.

The 19.3 antibody was evaluated as a potential capture reagent for Aβoligomers in combination with three different antibodies as detectionantibodies 19.3, 7305 (i.e., 20C2, U.S. Pat. No. 7,780,963, which isincorporated herein by reference in its entirety), and 82E1, followingtheir biotinylation, in a sandwich ELISA format. Biotinylated 19.3 wasexamined as a detection antibody and paired with 19.3 as the captureantibody, in a test of overlapping epitopes. The presence of overlappingepitopes would be indicative of an Aβ construct with multiple epitopes,which suggests the presence of a dimer or higher order Aβ oligomers. The19.3×19.3 overlapping epitope ELISA had a limit of detection (LoD) forAβ oligomers of 98 pg/mL. Sandwich ELISAs for the antibody pair 19.3 and82E1 had a LoD of 1.3 pg/mL and a lower limit of reliable quantification(LLoRQ) of 4.2 pg/mL for Aβ oligomers and the ratio of signal from Aβoligomers/Aβ monomer was approximately 1000:1, showing that the assaywas 1000 fold more selective for Aβ oligomers over Aβ40 monomer.Therefore, while both the capture and detection antibodies of the kitand method of this invention both recognize the N-terminal portion ofAβ1-42, in certain embodiments, the epitope of the capture and detectionantibody do not overlap or overlap by less than 3, 2, or 1 amino acidresidue.

To provide specificity for oligomers of Aβ, particular embodiments ofthis invention embrace the use of a capture antibody that recognizes alinear epitope and a conformational epitope. Such an antibody isdescribed herein as being selective or specific for Aβ oligomers. As isknown in the art, a conformational epitope is an epitope wherein theprimary sequence of the amino acids comprising the epitope is not thesole defining component of the epitope recognized. Typically aconformational epitope encompasses an increased number of amino acidsrelative to a linear epitope. With regard to recognition ofconformational epitopes, the antibody recognizes a three-dimensionalstructure of the peptide or protein. For example, when a proteinmolecule folds to form a three-dimensional structure, certain aminoacids and/or the polypeptide backbone forming the conformational epitopebecome juxtaposed enabling the antibody to recognize the epitope.Methods of determining conformation of epitopes include but are notlimited to, for example, x-ray crystallography, two-dimensional nuclearmagnetic resonance spectroscopy and site-directed spin labeling andelectron paramagnetic resonance spectroscopy. See, for example, EpitopeMapping Protocols in Methods in Molecular Biology (1996) Vol. 66, Morris(Ed.).

Preferably, a capture antibody that is selective for Aβ oligomer has ahigher affinity for Aβ1-42 oligomers or ADDLs than for Aβ1-42 monomer,Aβ1-40 monomer, plaques and/or amyloid beta fibrils. As demonstratedherein, selectivity can be assessed using a variety of methodsincluding, but not limited to competitive binding assays such asone-sided ELISA, sandwich ELISA or competitive ELISA assays. Using suchassays (Example 15 and FIG. 1), a number of antibodies, e.g., h3B3,14.2, 7.2, 11.4, 13.1, 17.1, 19.3, 20C2, 2A10, 2B4, 2D6, 5F10, 4E2, 4C2,and W0-2, were found to selectively bind oligomers over amyloid beta1-40 monomer and fibrils. Based upon this analysis, an antibody isdefined as being specific for Aβ oligomers if it exhibits at least a2-fold, 3-fold, 4-fold, 5-fold higher affinity for Aβ oligomers comparedto one or more of Aβ1-42 monomer, Aβ1-40 monomer, plaques or amyloidbeta fibrils when assessed in a conventional assay, e.g., BIACORE,KINEXA, or one-sided ELISA. In particular embodiments, the affinity ofthe capture antibody for Aβ1-42 oligomers compared to Aβ1′-40 monomersin a competitive binding assay is at least 500:1. In other embodiments,the affinity of the capture antibody for amyloid beta 1-42 oligomerscompared to amyloid beta 1-42 monomers in a sandwich ELISA assay is atleast 500:1, at least 600:1, at least 700:1, at least 800:1, at least900:1 or more preferably at least 1000:1.

In particular embodiments of this invention, variants of antibody h3B3(i.e., 14.2, 7.2, 11.4, 13.1, 17.1, 19.3), or variants of antibody 19.3(i.e., 19.3 N33S, 19.3 N33T, 19.3 N33A, 19.3 N33E, 19.3 N33D, 19.3N33S-N35Q, 19.3 N33S-N35S, 19.3 N33S-N35T, 19.3 N33S-N35A, 19.3 N58Q,19.3 N58S, 19.3 N58T, 19.3N35A) are used as the capture antibody in thekit and method of this invention. Accordingly, in some embodiments, acapture antibody of the kit and method of the invention has a lightchain variable region with a CDR1 having the sequenceArg-Ser-Ser-Gln-Ser-Ile-Val-His-Ser-Xaa₁-Gly-Xaa₂-Thr-Thy-Leu-Glu (SEQID NO:1), wherein Xaa₁ is Asn, Ser, Thr, Ala, Asp or Glu and Xaa₂ isAsn, His, Gln, Ser, Thr, Ala, or Asp, a CDR2 having the sequenceLys-Ala-Ser-Xaa₁-Arg-Phe-Ser (SEQ ID NO:2), wherein Xaa₁ is Asn, Gly,Ser, Thr, or Ala, and a CDR3 having the sequencePhe-Gln-Gly-Ser-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅ (SEQ ID NO:3), wherein Xaa₁ isArg, Lys or Tyr, Xaa₂ is Val, Ala, or Leu, Xaa₃ is Pro, His, or Gly,Xaa₄ is Ala, Pro, or Val, and Xaa₅ is Ser, Gly, or Phe; and a heavychain variable region with a CDR1 having the sequenceGly-Phe-Thr-Phe-Ser-Ser-Phe-Gly-Met-His (SEQ ID NO:4), a CDR2 having thesequenceTyr-Ile-Ser-Arg-Gly-Ser-Ser-Thr-Ile-Tyr-Tyr-Ala-Asp-Thr-Val-Lys-Gly (SEQID NO:5), and a CDR3 having the sequence Gly-Ile-Thr-Thr-Ala-Leu-Asp-Tyr(SEQ ID NO:6). Accordingly, in some embodiments, a capture antibody ofthe kit and method of the invention has a light chain variable regionwith a CDR1 having the sequenceArg-Ser-Ser-Gln-Ser-Ile-Val-His-Ser-Xaa₁-Gly-Xaa₂-Thr-Thy-Leu-Glu (SEQID NO:1), wherein Xaa₁ is Thr, Ala, Asp or Glu and Xaa₂ is Asn, His,Gln, Ser, Thr, Ala, or Asp or wherein Xaa₁ is Asn, Ser, Thr, Ala, Asp orGlu and Xaa₂ is Thr, a CDR2 having the sequenceLys-Ala-Ser-Xaa₁-Arg-Phe-Ser (SEQ ID NO:2), wherein Xaa₁ is Thr, and aCDR3 having the sequence Phe-Gln-Gly-Ser-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅ (SEQID NO:3), wherein Xaa₁ is Arg, Lys or Tyr, Xaa₂ is Val, Ala, or Leu,Xaa₃ is Pro, His, or Gly, Xaa₄ is Ala, Pro, or Val, and Xaa₅ is Ser,Gly, or Phe; and a heavy chain variable region with a CDR1 having thesequence Gly-Phe-Thr-Phe-Ser-Ser-Phe-Gly-Met-His (SEQ ID NO:4), a CDR2having the sequenceTyr-Ile-Ser-Arg-Gly-Ser-Ser-Thr-Ile-Tyr-Tyr-Ala-Asp-Thr-Val-Lys-Gly (SEQID NO:5), and a CDR3 having the sequence Gly-Ile-Thr-Thr-Ala-Leu-Asp-Tyr(SEQ ID NO:6) and such a capture antibody also forms part of the presentinvention.

In some embodiments, the capture antibody of the kit and method of theinvention is a variant of antibody h3B3 (i.e., 14.2, 7.2, 11.4, 13.1,17.1, 19.3). In accordance with this embodiment, the capture antibodyhas a light chain variable region with a CDR1 having the sequenceArg-Ser-Ser-Gln-Ser-Ile-Val-His-Ser-Asn-Gly-Asn-Thr-Tyr-Leu-Glu (SEQ IDNO:14), a CDR2 having the sequence Lys-Ala-Ser-Asn-Arg-Phe-Ser (SEQ IDNO:15), and a CDR3 of SEQ ID NO:3; and a heavy chain variable regionwith a CDR1 of SEQ ID NO:4, a CDR2 of SEQ ID NO:5, and a CDR3 of SEQ IDNO:6.

In other embodiments, the capture antibody of the kit and method of theinvention is a variant of antibody 19.3, wherein the CDR1 of the lightchain variable region has been mutated (i.e., 19.3 N33S, 19.3 N33T, 19.3N33A, 19.3 N33E, 19.3 N33D, 19.3 N33S-N35Q, 19.3 N33S-N35S, 19.3N33S-N35T, 19.3 N33S-N35A). In accordance with this embodiment, thecapture antibody has a light chain variable region with a CDR1 of SEQ IDNO:1, a CDR2 of SEQ ID NO:15, and a CDR3 having the sequencePhe-Gln-Gly-Ser-Arg-Leu-Gly-Pro-Ser (SEQ ID NO:16); and a heavy chainvariable region with a CDR1 of SEQ ID NO:4, a CDR2 of SEQ ID NO:5, and aCDR3 of SEQ ID NO:6.

In still other embodiments, the capture antibody of the kit and methodof the invention is a variant of antibody 19.3, wherein the CDR2 of thelight chain variable region has been mutated (i.e., 19.3 N58Q, 19.3N58S, 19.3 N58T, 19.3N35A). In accordance with this embodiment, thecapture antibody has a light chain variable region with a CDR1 of SEQ IDNO:14, a CDR2 of SEQ ID NO:2, a CDR3 of SEQ ID NO:16; and a heavy chainvariable region with a CDR1 of SEQ ID NO:4, a CDR2 of SEQ ID NO:5, and aCDR3 of SEQ ID NO:6.

In certain embodiments, the CDR1 of the light chain variable region ofthe capture antibody has the sequenceArg-Ser-Ser-Gln-Ser-Ile-Val-His-Ser-Xaa₁-Gly-Xaa₂-Thr-Thy-Leu-Glu (SEQID NO:1), wherein Xaa₁ is Thr, Ala, Asp or Glu and Xaa₂ is Thr. In otherembodiments, the CDR2 of the light chain variable region of the captureantibody has the sequence Lys-Ala-Ser-Xaa₁-Arg-Phe-Ser (SEQ ID NO:2),wherein Xaa₁ is Thr.

To facilitate production and enhance storage and use of the captureantibody in the kit and method of this invention, certain embodimentsinclude the use of a capture antibody that exhibits less than a 10-folddecrease in EC₅₀, in an ELISA-based assay with Aβ oligomers, when storedat 40° C. for 1 month. More preferably, the capture antibody exhibitsless than a 6-fold, 5-fold, 4-fold, 3-fold, or 2-fold decrease in EC₅₀when stored at 40° C. for 1 month. Antibody stability can be assessed asdescribed in the Examples herein. Antibodies having such stability atelevated temperatures are provided in Examples 7 and 9.

While the detection antibody recognizes a linear epitope located in theN-terminus of Aβ1-42, said antibody may or may not also bind aconformational epitope. In this respect, there are number of antibodiesof use as the detection antibody in the kit and method of thisinvention. As provided in Table 1, any one of antibodies 6E10, BAM-10,W0-2, 26D6, 2A10, 2B4, 4C2, 4E2, 2H4, 20C2, 2D6, 5F10, 1F4, 1F6, 2E12,3B3 or 82E1 recognize a linear epitope located in the N-terminus ofAβ1-42 and is therefore of use in the kit and method of the invention.In certain embodiments, the detection antibody binds a 5 to 10 aminoacid residue N-terminal epitope of Aβ1-42 having the sequence DAEFR (SEQID NO:13). In particular embodiments, the detection antibody is 82E1.

To facilitate detection of a capture antibody/Aβ oligomer/detectionantibody complex, certain embodiments include the use of a labeleddetection antibody. A variety of labels are well-known in the art andcan be adapted to the practice of this invention. For example,fluorescent labels, luminescent labels and light-scattering labels(e.g., colloidal gold particles) have been described. See, e.g., Csakiet al. (2002) Expert. Rev. Mol. Diagn. 2:187-93.

Fluorescent labels of use in this invention include, but not limited to,hydrophobic fluorophores (e.g., phycoerythrin, rhodamine, ALEXA FLUOR488, ALEXA FLUOR 546 and fluorescein), green fluorescent protein (GFP)and variants thereof (e.g., cyan fluorescent protein and yellowfluorescent protein), and quantum dots. See e.g., Haughland (2003)Handbook of Fluorescent Probes and Research Products, Ninth Edition orWeb Edition, from Molecular Probes, Inc., or The Handbook: A Guide toFluorescent Probes and Labeling Technologies, Tenth Edition or WebEdition (2006) from Invitrogen for descriptions of fluorophores emittingat various different wavelengths. For use of quantum dots as labels forbiomolecules, see e.g., Dubertret, et al. (2002) Science 298:1759;Nature Biotech. (2003) 21:41-51. In particular embodiments, the label isa hydrophobic fluorophores such as an ALEXA FLUOR.

Labels can be introduced onto the detection antibody by techniquesestablished in the art. For example, kits for fluorescently labelingantibodies with various fluorophores are available from Invitrogen Corp.Similarly, signals from the labels (e.g., absorption by and/orfluorescent emission from a fluorescent label) can be detected by thetechniques exemplified herein (i.e., ENVISION or ERENNA system, whereinthe fluorescent tagged detecting antibody is uncoupled from the sandwichELISA complex and subsequently detected) or by essentially any methodknown in the art. For example, multicolor detection, detection of FRET,fluorescence polarization, and the like, are well-known in the art. Forexample, flow cytometers are widely available, e.g., fromBecton-Dickinson and Beckman Coulter and LUMINEX 100 and LUMINEX HTSsystems are available from Luminex Corporation.

To enhance the sensitivity of the kit and method of this invention forAβ oligomers, one embodiment of this invention includes the use of ameans or substrate for concentrating an antibody-antigen complex. Asdemonstrated herein, the performance of two antibody pairs was assessedin a paramagnetic microparticle detection system, specifically theERENNA system (SINGULEX, Almeda, Calif.), employing detection of afluorescent tagged detecting antibody that is uncoupled from thesandwich ELISA complex. Performance of a 19.3×82E1 sandwich ELISA wasimproved such that the 19.3×82E1 antibody pair enabled detection of Aβoligomer signals in AD CSF samples at higher levels compared to eitherage-matched or younger control samples. More specifically, the assay LoDimproved approximately thirty-fold, to 0.04 pg/mL, while the LoRQimproved ten-fold, to 0.42 pg/mL. Similarly, the Aβ oligomer/Aβ monomerratio was also improved, to 5000:1. Therefore, a means or substrate forconcentrating an antibody-antigen complex can be used to increase thesensitivity of the kit and method of this invention.

Substrates for concentrating antigen-antibody complexes are known in theart and include, but are not limited to solid surfaces (e.g., beads),fluorescent polymeric beads, magnetic beads, which can be bonded orattached to the capture antibody.

Solid surfaces, such as beads, can be bound to the capture antibody,such that the capture antibody/Aβ oligomer/detection antibody complexcan be concentrated by centrifugation or filtration. Fluorescent beadscan be prepared, for example, by embedding or covalently coupling afluorescent dye onto a polymeric particle and attaching the particle tothe capture antibody. The fluorescent microparticles can be analyzedmanually or by other methods known in the art but preferably using anautomated technique, e.g., flow cytometry, such as disclosed in U.S.Pat. No. 4,665,024. The versatility of the fluorescent particles can befurther enhanced by the incorporation of multiple fluorescent materialsin a single particle. Magnetic particles, including, paramagnetic andsuperparamagnetic can alternatively be used to concentrateantibody-antigen complexes via a magnetic field. Such particles areknown in the art and, in addition to their magnetic properties (i.e.magnetic, paramagnetic, and superparamagnetic), can be classified, forexample, into three broad categories based on their relative descendingsize: magnetic particulate labels, colloidal magnetic labels, andmolecular magnetic labels, see for example U.S. Pat. No. 6,412,359. Incertain embodiments, capture antibody is bound to a magneticmicroparticle as described in the method described herein.

Using the kit and method of this invention, it has been demonstratedthat the level of detection of amyloid beta 1-42 oligomers is less than5 pg/mL. Indeed, using two anti-Aβ oligomer antibodies, 19.3 and 82E1,along with paramagnetic micro-particle detection, in a sandwich ELISAassay, it has now been shown that Aβ oligomers can be detected in abiological sample to a limit of detection of 40 fg/mL. Accordingly, insome embodiments of this invention, the limit of detection of the kitand method of the invention is less than 5 pg/mL, less than 3 pg/mL,less than 1 pg/mL, less than 500 fg/mL, or less than 100 fg/mL. Incertain embodiments, the limit of detection of the kit and method of theinvention is in the range of 40 fg/mL and 5 pg/mL.

The term “limit of detection” of “LoD,” as used herein, refers to thesensitivity of the assays at the lowest concentration that can bedetected above a sample which is identical except for the absence of theAβ oligomers. The signal in the absence of Aβ oligomers is defined asthe “Background.” As used herein, the LoD for Aβ oligomers was definedas ≧3 standard deviations above the mean of the background. The “lowerlimit of reliable quantification” or “LLoRQ,” as used herein, refers tothe sensitivity of the assay in combination with the coefficient ofvariability to indicate the lowest concentration that can be reliablyand reproducibly differentiated from background. This limit typicallydefines the practical working range of the assay at the low end ofsensitivity and is the concentration that delivers a coefficient ofvariability of ≦20% across ≧ three measured values.

While Aβ oligomers have been found in biological samples, particularlyin CSF (Georganopoulou, at al. (2005) supra; Klyubin, et al. (2008)supra), the limits associated with known detection methods (includingboth sensitivity and selectivity) have not enabled this level ofreliable detection, let alone, quantification of Aβ oligomers for use toclassify the disease state of the patient or for the development of ADtherapeutics. In contrast, using the method of this invention, highlysignificant elevations in Aβ oligomers were demonstrated in clinicallyconfirmed AD samples as compared to either young or age-matchedcontrols. These same samples were used to measure levels of Aβ1-42 andAβ1-40 monomer and confirmed that in the AD samples Aβ1-42 monomer wassignificantly reduced as compared to the controls, while the Aβ1-40monomer levels were unchanged. The Aβ oligomer sandwich ELISA assaydemonstrated significant correlations between Aβ oligomer concentrationand performance on a cognitive test widely used to measure AD severity,known as the Mini-Mental State Exam (MMSE); the higher the cognitivescore (up to a value of 30, which is cognitively normal) the lower thelevel of Aβ oligomer in the CSF. Accordingly, in some embodiments ofthis invention, the method and kit are of use in confirming a diagnosisor diagnosing AD or AD severity. In addition, the kit and method of thisinvention can be used to identify patients at an early stage of disease(i.e., a prognostic assay).

Therefore, this invention also provides a method for detecting oligomersof Aβ. In accordance with this method, a biological sample havingoligomers of Aβ is obtained from an animal, preferably a human; thebiological sample is contacted with a capture antibody, as describedherein, under conditions sufficient to form a capture antibody/oligomerof Aβ complex; the capture antibody/oligomer of Aβ complex is thendetected using a detection antibody, as described herein. In otherembodiments of this method, a biological sample from an animal,preferably a human, is contacted with a capture antibody, as describedherein, under conditions sufficient to form a capture antibody/oligomerof Aβ complex; the capture antibody/oligomer of Aβ complex is thendetected using a detection antibody, as described herein. The term“biological sample” or “fluid sample,” as used herein, refers to anytype of fluid, as compared to a tissue, or a vertebrate. Typicalexamples that may be used in the assays herein are blood, urine, tears,saliva, and cerebrospinal fluid, which is used in one embodiment of theinvention. All other kinds of body fluids may also be used if Aβoligomers are present. However, in particular embodiments, thebiological sample is CSF.

This method is a sensitive and selective competitive binding assay, suchas a sandwich ELISA assay, which detects and quantifies endogenous Aβoligomers in biological samples such as CSF samples from both AD andhuman control individuals. While Alzheimer's disease or AD isparticularly described herein as one condition that can be diagnosedusing the kit and method of this invention, the spectrum of dementias orcognitive impairment resulting from neuronal degradation associated withthe formation of Aβ oligomers or formation or deposition of Aβ plaquesor neurofibrillar tangles includes, but not limited to, Down's Syndrome,Lewy body dementia, Parkinson's disease, preclinical Alzheimer'sdisease, mild cognitive impairment due to Alzheimer's disease, earlyonset Alzheimer's disease (EOD), familial Alzheimer's disease (FAD),thru the advance cognitive impairment of dementia due to Alzheimer'sdisease (Jack, et al. (2011) Alzheimer's Dement. 7(3):257-262), anddiseases associated with the presence of the ApoE4 allele. Therefore,the kit and method can be used in the diagnosis of any one of thesediseases or conditions.

The invention is described in greater detail by the followingnon-limiting examples.

Example 1 Aβ Preparations

Aβ1-40 and Aβ1-42 (amyloid β peptide 1-40, amyloid β peptidel-42) wereobtained from the American Peptide Co. (Sunnyvale, Calif.).

Monomer Preparation. To generate monomer preparations, Aβ1-40 or Aβ1-42was dissolved in 1,1,1,3,3,3 hexafluoro-2-propanol (HFIP; Sigma-Aldrich,St. Louis, Mo.) to eliminate any pre-existing secondary structure thatcould act as a “seeds” for aggregation. The HFIP was removed byevaporation to form an Aβ1-40 or Aβ1-42 peptide film. Room temperatureAβ1-40 or Aβ1-42 peptide film was dissolved in 2 mL of 25 mM boratebuffer (pH 8.5) per mg of peptide, divided into aliquots, and frozen at−70° C. until used.

ADDL Preparation. The Aβ42 peptide film (1 mg Aβ42 dried down from 100%HFIP solvent) was dissolved in 44 μL of DMSO, to which 1956 μl of coldF12 media (GIBCO®, Invitrogen, Carlsbad, Calif.) was added with gentlemixing. The mixture was incubated at room temperature for 18 to 24hours. Samples were centrifuged at 14,200 g for 10 minutes at roomtemperature. Supernatant was transferred to a fresh tube and wasfiltered through 0.5 ml column YM-50 filter tube (Millipore, BedfordMass.; 0.5 ml) via centrifugation at 4,000 rpm for 15 minutes at 4° C.The retentate was collected by reversing the filter insert, replacedinto a new collection tube, and centrifuged at 4,000 rpm for 5 minutesat 4° C. Protein concentration was measured pre-filtration by BradfordAssay (BioRad, Hercules, Calif.) and reported as μM (calculated based onAβ monomer molecular weight (MW 4513)). All samples were stored at −80°C. until used.

Biotinylated ADDLs (bADDLs) Preparation. bADDLs were prepared using thesame method as described for ADDLs, with N-terminal biotinylated Aβ 1-42peptide (American Peptide, Sunnyvale, Calif.) as the starting material.

Fibril Preparation. The fibril preparations were made by adding 2 mL of10 mM hydrochloric acid per mg of Aβ1-42 peptide film. The solution wasmixed on a vortex mixer at the lowest possible speed for five to tenminutes and the resulting preparation was stored at 37° C. for 18 to 24hours before use.

Example 2 Preparation of Affinity-Matured 3B3 Antibodies

Panning Humanized Antibody Library. An affinity mature library of ahumanized anti-ADDL antibody, h3B3, (See U.S. Pat. Nos. 7,811,563 and7,780,963) was constructed in which part of the light chain CDR3 aminoacid sequence was subjected to random mutagenesis. To cover the entireCDR3 region, two sub-libraries were built. One library was composed ofthe parental heavy chain variable region and mutated amino acids in theleft half of the light chain CDR3 and the other in the right half of thelight chain CDR3. A similar strategy was used for heavy chain CDRsrandom mutagenesis with three sub-libraries.

Humanized 3B3 was subjected to affinity maturation using methods knownin the art. The h3B3 variable regions were cloned in a Fab displayvector (pFab3D). In this vector, the variable regions for heavy andlight chains were inserted in-frame to match the CH1 domain of theconstant region and the kappa constant region, respectively. In Fab3D,myc epitope and six consecutive histidine amino acid residues follow theCH1 sequence, which is then linked to the phage pIII protein fordisplay. All positions in the heavy and light chain CDR3s were randomlymutagenized using degenerate oligonucleotide sequences incorporated intothe PCR primers. To accommodate the physical size, the sub-librarieswere constructed with each focusing on 5-6 amino acid residues. Thevector DNA of h3B3 was used as template DNA to amplify both heavy andlight chains with the mutated PCR primers (Table 2). After PCRamplification, the synthesized DNA fragments were separated on a 1.3%agarose gel, the primers removed and the variable fragments digestedwith restriction enzymes: BsiWI and XbaI cloning sites for light chainvariable cloning, XhoI and ApaI for heavy chain variable cloning.

TABLE 2 3B3 Affinity SEQ Maturation ID Library Primer Primer SequenceNO: Light Chain Forward tatggcttctagagatgtggtgatg 17 Libraries Reversetgcagccaccgtacgcttgatctcc 18 agcttggtgccctggccaaaggtggggggcacmnnmnnmnnmnnmnngca gtagtag tgcagccaccgtacgcttgatctcc 19agcttggtgccctggccaaamnnmn  nmnnmnnmnngctgccctgg Heavy Chain Forwardaggcggccctcgaggaggtgcagc 20 Libraries Reverse agaccgatgggcccttggtggaggc21 gctggacacggtcaccagggtgccc tggccccamnnmnnmnnmnnmnngg tgatgcccagaccgatgggcccttggtggaggc 22 gctggacacggtcaccagggtgccctggccccagtagtccagmnnmnnmn nmnnmnnccgggcacag M = A/C, N = A/C/G/T.

To construct an affinity maturation library in pFab3D phage displayvector, pFab3D-3B3 DNA was digested with the same pair of therestriction enzymes, purified and the PCR fragments for heavy or lightchain variables ligated with T4 ligase (Invitrogen, Carlsbad, Calif.)overnight at 16° C. The ligation products were then transfected into E.coli TG1 electroporation-competent cells (Stratagene, AgilentTechnologies, Santa Clara, Calif.) and aliquots of the bacterial cultureplated on LB agar-carbenicillin (50 μg/mL) plates to titer library size.The remaining cultures were either plated on a large plate withcarbenicillin and incubated at 30° C. overnight for E. coli librarystock or infected with helper phage M13K07 (Invitrogen, Carlsbad,Calif.; 10¹¹ pfu/mL) by incubating at room temperature and 37° C. forten minutes. Subsequently, 2TY medium with carbenicillin (50 μg/mL) wasadded and the culture was incubated at 37° C. for one hour with shaking.Kanamycin (70 μg/ml) was then added and the cultures grown overnight at30° C. with shaking. The phage culture supernatant was titered andconcentrated by precipitation with 20% (v/v) PEG (polyethyleneglycol)/NaCl, resuspended in PBS, sterilized with a 0.22 μm filter, andaliquots made for phage library panning.

The phage library panning was then conducted as summarized in Table 3.

TABLE 3 Panning Rounds Round 1 Round 2 Round 3 Round 4 Antigen 180 nM 60nM 20 nM 10 nM concentration

Input phage from the Fab display phage libraries (100 μl, about 10¹¹⁻¹²pfu) were blocked with 900 μL of blocking solution (3% non-fat dry milkin PBS) to reduce nonspecific binding to the phage surface.Streptavidin-coated beads were prepared by collecting 200 μL of the beadsuspension in a magnetic separator and removing supernatants. The beadswere then suspended in 1 mL of blocking solution and put on a rotarymixer for 30 minutes. To remove non-specific streptavidin binding phage,the blocked phage library was mixed with the blocked streptavidin-coatedbeads and placed on a rotary mixer for thirty minutes. Phage suspensionsfrom the de-selection process were transferred to a new tube and 200 μLof antigen, 10% bADDL, was added and incubated for two hours forantibody and antigen binding. After the incubation, the mixture wasadded into the blocked streptavidin-coated beads and incubated on arotary mixer for one hour to capture the antibody/antigen complex onstreptavidin beads. The beads with captured 10% bADDL/phage complexeswere washed five times with PBS/0.05% TWEEN 20 and then twice with PBSalone. The bound phages were eluted from the bADDL with 200 μL of 100 mMTEA and incubated for twenty minutes. The eluted phage were thentransferred to a 50 mL tube, neutralized with 100 μL of 1 M Tris-HCl, pH7.5, and added to 10 mL of E. coli TG1 cells with an OD600 nm between0.6-0.8. After incubation at 37° C. with shaking for one hour, culturealiquots were plated on LB agar-carbenicillin (50 μg/mL) plates to titerthe output phage number, and the remaining bacteria centrifuged andsuspended with 500 μl 2xYT medium (Teknova, Hollister, Calif.), platedon bioassay YT agar plates (Teknova, Hollister, Calif.) containing 100μg/ml ampicillin and 1% glucose. The bioassay plates were grownovernight at 30° C.

After each round of panning, single colonies were randomly picked toproduce phage in 96-well plates. The procedure for phage preparation in96-well plates was similar to that described above except no phageprecipitation step was used. Culture plates containing colonies growingin 120 μL of 2xYT medium (16 g BACTO-tryptone, 10 g BACTO-yeast extract,5 g NaCl (all BD Biosciences, Franklin Lakes, N.J.), ddH₂O to 1 L) with100 μg/ml ampicillin and 0.1% glucose were incubated overnight in aHIGRO shaker (Genomic Solutions, Ann Arbor, Mich.) at 30° C. withshaking at 450 rpm. The phage supernatants (about 100 μL) were directlyused for analysis in the ADDL binding ELISA. Binding of phage to ADDLswas detected with an anti-M13-antibody conjugated to horseradishperoxidase (HRP) (Amersham Bioscience, GE Healthcare, Waukesha, Wis.).

Example 3 Selection of Affinity Matured 3B3 Antibodies

From the light chain affinity maturation effort, a panel of seven clones(11.4, 17.1, 14.2, 13.1, 19.3, 7.2 and 9.2) showed strong bindingactivities to ADDLs when compared with h3B3 in a phage/Fab ELISA. Table4 shows the amino acid similarity for the clones selected from the lightchain affinity maturation library relative to parental antibody, h3B3.

TABLE 4 h3B3- humanized Antibody 11.4 17.1 14.2 13.1 19.3 7.2 9.2 LC11.4 — 98 98 96 96 96 97 97 17.1 — — 98 96 97 96 97 97 14.2 — — — 96 9798 98 98 13.1 — — — — 97 97 97 96 19.3 — — — — — 96 97 96 7.2 — — — — —— 98 96 9.2 — — — — — — — 97

Table 5 summarizes the amino acid sequences in CDR3 of the light chain(LC) of the selected clones compared to the CDR3 of the light chain forthe parental antibody, h3B3.

TABLE 5 Antibody LC-CDR3 Sequence SEQ ID NO: h3B3 (parental) FQGSHVPPT23 19.3 FQGSRLGPS 16 17.1 FQGSRVPAS 24 14.2 FQGSRVPPG 25 13.1 FQGSKAHPS26 7.2 FQGSYAPPG 27 9.2 FQGSRAPPF 28 11.4 FQGSRVPVR 29

Table 6 provides the sequence of a portion (positions 21-117) of thelight chain variable regions (LCVR) for the selected clones and theparental antibody, h3B3. CDR3 of each clone is shown in bold.

TABLE 6 Ab LCVR Sequence SEQ ID NO: h3B3PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSG 30VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPPTFGQGT KLEIK 19.3PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSG 31VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSRLGPSFGQGT KLEIK 17.1PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSG 32VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSRVPASFGQGT KLEIK 14.2PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSG 33VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSRVPPGFGQGT KLEIK 13.1PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSG 34VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSKAHPSFGQGT KLEIK 7.2PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSG 35VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSYAPPGFGQGT KLEIK 9.2PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSG 36VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSRAPPFFGQGT KLEIK 11.4PASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKASNRFSG 37VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSRVPVRFGQGT KLEIK

Table 6 Example 4 IgG Conversion of Affinity Matured 3B3 Antibodies

The seven leading Fab clones (11.4, 17.1, 14.2, 13.1, 19.3, 7.2 and 9.2)were selected for IgG conversion. The converted IgGs were expressedusing plasmid-based vectors. The expression vectors were built such thatthey contained all the necessary components except the variable regions.In the basic vectors, the expression of both light and heavy chains wasdriven by human CMV promoter and bovine growth hormone polyadenylationsignal. For the seven clones selected for IgG conversion, the heavychain variable region was in-frame fused with a human IgG2 heavy chainconstant region (SEQ ID NOs:38 and 39), while the light chain variableregion was in-frame fused with the kappa light chain constant region(SEQ ID NOs:40 and 41). The heavy (SEQ ID NOs:42 and 43) and light (SEQID NOs:44 and 45) chain leader sequences, which mediate the secretion ofthe antibodies into the culture media, were also in-frame fused with thevariable regions accordingly. For the heavy chain expression vectors,the constant region could be selected from a different subclass isotype,e.g., IgG1 or IgG2. Between the leader sequence and the constant region,the intergenic sequences contain cloning sequences for seamless in-framefusion of the incoming variable region with the leader sequence at its5′-end and the constant region at its 3′-end using an IN-FUSION cloningstrategy (Clontech, Mountain View, Calif.). The IN-FUSION Dry-Down PCRCloning Kit (Clontech, Mountain View, Calif.) was used for PCRamplification of the variable regions. The dry-down cloning kitcontained all the necessary components for PCR reaction. PCR primers andtemplate DNAs were added. The expression vectors carry oriP from the EBVviral genome. The oriP/EBNA1 pair is often used to prolong the presenceof the expression vector inside the transfected cells and widely usedfor the extension of the expression duration (Lindner, et al. (2007)Plasmid 58:1-12) for prolonged expression in 293EBNA cells, bacterialsequences for a kanamycin selection marker, and a replication origin inE. coli. When the variable regions were inserted, the IgGs were directlyexpressed in mammalian cells. All heavy chain variable regions hereinwere cloned into an IgG1 expression vector (pVl JNSA-BF-HCG1) and thelight chain variable regions were cloned into a matching kappa or lambdaexpression vector (pVl JNSA-GS-FB-LCK).

Example 5 Affinity Matured 3B3 Antibody Cloning and Expression

The seven leading clones (11.4, 17.1, 14.2, 13.1, 19.3, 7.2 and 9.2)were produced as monoclonal antibodies and purified for furthercharacterization. The cloning procedure for the resulting antibodyexpression vectors was as follows. The variable regions werePCR-amplified, wherein the PCR reactions were carried out in a volume of25 μL containing high fidelity PCR master mix, template (1 μL), andforward and reverse primers (1 μL each). PCR conditions: 1 cycle of 94°C., 2 minutes; 25 cycles of 94° C., 1.5 minutes; 60° C., 1.5 minutes;72° C., 1.5 minutes and 72° C., 7 minutes; 4° C. until removed. The PCRproducts were then digested with DpnI and purified with QIAQUICK platekit (Qiagen, Venlo, The Netherlands). One hundred nanograms of thecorresponding previously linearized heavy chain or light chain vectorswas annealed to 10 ng of the PCR fragment with an IN-FUSION reaction(IN-FUSION Dry-Down Cloning Kit, Clontech, Mountain View, Calif.). Thereaction mixture was transformed to XL2 Blue MRF′ competent cells andplated overnight on agar plates containing 50 pg/mL kanamycin. Lightchain constructs were digested with HindIII+NotI and heavy chainconstructs were digested with AspI+HindIII to check structure byrestriction analysis. The DNA sequences for all the clones wereconfirmed by sequence analysis.

Sequencing confirmed constructs of light chain and heavy chain DNA weretransfected in 293 FREESTYLE cells (Invitrogen, Carlsbad, Calif.). The293 FREESTYLE cells were transfected using 293 Transfectin (Invitrogen,Carlsbad, Calif.). EBNA monolayer cells were transfected usingpolyethylenimine-based transfection reagents. Transfected cells wereincubated at 37° C./5% CO₂ for seven days in OPTI-MEM serum-free medium(Invitrogen, Carlsbad, Calif.). The medium was collected, centrifuged,filtered through 0.22 μm filtration system (Millipore, Billerica,Mass.), and then concentrated by a CENTRICON centrifuge filter(Millipore, Billerica, Mass.). Concentrated medium was mixed 1:1 withbinding buffer (Pierce, Thermo Fisher Scientific, Rockford, Ill.), andsubsequently loaded onto a pre-equilibrated protein A/G column (Pierce,Thermo Fisher Scientific, Rockford, Ill.) or HI-TRAP rProtein A FF (GEHealthcare, Waukesha, Wis.). The loaded column was washed with bindingbuffer and eluted with elution buffer (Pierce, Thermo Fisher Scientific,Rockford, Ill.). Eluted antibody was neutralized immediately anddialyzed against PBS buffer for overnight. Dialyzed antibody wasconcentrated with an AMICON centrifuge filter (Pierce, Thermo FisherScientific, Rockford, Ill.) and protein concentration was determined atOD280 nm with the extinct coefficient of 1.34 mg/mL. Purified antibodywas analyzed using SDS-PAGE (Invitrogen, Carlsbad, Calif.), or proteinLABCHIP (Caliper LifeSciences, Hopkinton, Mass.). SDS-PAGE was run undernon-reducing conditions.

Example 6 Characterization of Affinity Matured 3B3 Antibodies

ELISA. The selected anti-ADDL antibodies, i.e., those derived from theparental antibody, h3B3, where first assessed in a three-pronged AβELISA to evaluate binding of the antibody to monomer Aβ, ADDLs, andfibrillar A. Polyclonal anti-ADDLs IgG (M90/1; Bethyl Laboratories,Inc., Montgomery, Tex.) was plated at 0.25 mg/well on IMMULON REMOVAWELLstrips (Dynatech Labs, Chantilly, Va.) for 2 hours at room temperatureand the wells blocked with 2% BSA in TBS. Samples (monomeric Aβ, ADDLs,or fibrillar Aβ) diluted with 1% BSA in F12 were added to the wells,allowed to bind for 2 hours at 4° C., and washed 3× with BSA/TBS at roomtemperature. Monoclonal antibodies diluted in BSA/TBS were incubated for90 minutes at room temperature and detected with a VECTASTAIN® ABC kitto mouse IgG. The HRP label was visualized with BIO-RAD peroxidasesubstrate and read at 405 nm on a Dynex MRX-TC microplate reader.

As shown in FIG. 1, with the exception of antibody 9.2, all of theanti-ADDL antibodies showed preferential binding to ADDLs relative toh3B3, selective (Comp 1 and 3: bind only ADDLs), non-selective (Comp 2:bind all forms of Aβ evaluated) comparators, and a control (noantibody). Antibody 9.2 showed low binding to all forms of Aβ, whichsuggested that its binding affinity was adversely affected during IgGconversion and/or antibody production. A summary of the ratio ofADDL:monomer and ADDL:fibrillar binding of the antibodies in this assayis presented in Table 7.

TABLE 7 Antibody ADDL:Monomer ADDL:Fibrillar h3B3 3.2 2.2 14.2 4.2 2.37.2 3.2 2.1 11.4 2.4 2.4 9.2 4.0 0.5 13.1 2.4 2.0 17.1 3.2 2.1 19.3 2.52.0

A full titration curve was generated for each antibody and h3B3 todetermine their binding affinity for ADDLs, as compared with monomer Aβ.Biotinylated ADDLs (50 pmol/well) or monomer Aβ1-40 (100 pmol/well) wereadded to a high-capacity streptavidin-coated plate (Sigma-Aldrich, St.Louis, Mo.) and incubated for two hours at room temperature. The plateswere washed in PBS with 0.05% TWEEN (six times) and then PBS alone(three times) prior to blocking wells with 5% non-fat dry milk in PBSfor one hour at room temperature. The wells were then washed and aserial dilution of antibody samples added to the plates and allowed tobind for two hours at room temperature. After incubation and washing,the antibody binding was detected with a goat anti-human IgG-Fcsecondary antibody conjugated to horse radish peroxidase (HRP) (1:1000;one hour at room temperature). The HRP label was visualized withtetramethyl benzidine (Virolabs, Chantilly, Va.) as a substrate and readat 450 nm on a microplate reader. This analysis confirmed that six ofthe seven affinity-matured antibodies showed preferential binding toADDLs. See FIG. 2, which compares the preferential binding of h3B3 and19.3 for ADDLs over monomeric Aβ1-40.

Cell-Based Binding Assay. It has been shown that some anti-ADDLantibodies having preferential binding to ADDLs but cannot prevent ADDLbinding to primary hippocampal neurons (Shughrue, et al. (2010)Neurobiol. Aging 31:189-202). To demonstrate that the anti-ADDLantibodies could block ADDL binding to neurons, a cell-based bindingassay was carried out. Anti-ADDL antibodies were mixed with 500 nMbADDLs, with the final antibody concentrations ranging from 1.8 nM to450 nM. As a control, the same concentration of heat-denatured antibody(98° C. for minutes) was mixed with bADDLs. The antibody-bADDL mixtureswere incubated in siliconized microcentrifuge tubes (Fischer Scientific,Pittsburgh, Pa.) at 37° C. for one hour with constant end-to-endrotation at a low speed. The mixtures were then applied to primaryhippocampal and/or cortical cultures and incubated at 37° C. for onehour. The incubation was terminated by removing the culture medium.Cells were subjected to fixation and post-fixation treatments. Cellswere then incubated with streptavidin conjugated with alkaline phosphate(AP) at 4° C. overnight, washed five times with PBS and reacted with theTROPIX CDP-Star chemiluminescent substrate (Life Technologies, Carlsbad,Calif.) at room temperature for 30 minutes. The bADDL binding intensitywas measured and recorded with an ENVISION microplate reader(PerkinElmer, Waltham, Mass.).

The results of this study showed that the anti-ADDL antibodies herein,specifically antibody 19.3, dramatically reduced ADDL binding to neurons(FIG. 3). However, a marked reduction in antibody activity in this assaywas observed when the antibodies were heat-denatured (FIG. 3).

In Vitro FcRn Binding of Anti-ADDL Antibodies. To characterize theability of anti-ADDL antibodies to bind and dissociate immobilized humanFcRn, the seven h3B3 variant anti-ADDL antibodies were evaluated in aBIACORE FcRn binding assay, a surrogate system used to evaluate antibodyPK and predict the terminal half-life (t_(1/2)) of antibodies innon-human primates. Briefly, purified human FcRn protein was immobilizedonto a BIACORE CM5 biosensor chip and PBSP (50 mM NaPO₄, 150 mM NaCl and0.05% (v/v) TWEEN 20) pH 7.3 was used as running buffer. The monoclonalantibodies were diluted with PBSP, pH 6.0, to 100 nM, allowed to bindFcRn for 3 minutes to reach equilibrium and dissociated in pH 7.3running buffer. A report point (Stability) was inserted at 5 seconds atthe end of monoclonal antibody binding and the “% bound” was calculatedas RU_(stability)/RU_(binding) (%). This analysis indicated thatmonoclonal antibodies (mAbs) with identical Fc sequences but differentFab domains can bind and dissociate from FcRn with considerabledifferences.

A comparison was made of the seven h3B3 variant anti-ADDL antibodies,along with h3B3, two ADDL preferring antibodies (Comp 1 and 3) and anon-selective (Comp 2: binds all Aβ forms evaluated) comparator in theFcRn binding assay. A sensorgram was generated (FIG. 4) showing theinitial binding of the antibody at pH 6.0 and then the dissociation ofthe antibody at pH 7.3 from 180 seconds. As shown in FIG. 4, there was anoticeable difference between h3B3 and the other antibodies assessed.While h3B3 had a high percent bound to FcRn, the seven anti-ADDLantibodies of the present invention, as well as the two comparatorantibodies exhibited considerably lower binding.

Example 7 Binding Affinity of Anti-ADDL Antibody 19.3

Affinity-matured antibody 19.3 was selected for furthercharacterization. The complete DNA sequence and the deduced amino acidsequence for the variable region of the light chain was determined.BIACORE (GE Healthcare, Waukesha, Wis.) and KINEXA (Sapidyne, Boise,Id.) analyses were carried out to ascertain the binding affinity ofanti-ADDL antibody 19.3 for ADDLs and determine the selectivity of 19.3for ADDLs versus monomer Aβ. BIACORE- and KINEXA-based technologies arewidely used for the measurement of binding affinity betweenmacromolecules such as antibody and protein target.

BIACORE. In the Surface Plasmon Resonance (SPR) technology on whichBIACORE is based, quantitative measurements of the binding interactionbetween one or more molecules are dependent on the immobilization of atarget molecule to the sensor chip surface. Binding partners to thetarget can be captured as they pass over the chip. SPR detects changesin mass in the aqueous layer close to the sensor chip surface bymeasuring changes in refractive index. When molecules in the testsolution bind to a target molecule, the mass increases (k_(a)), whenthey dissociate the mass falls (k_(d)). This simple principle forms thebasis of the sensorgram, i.e., a continuous, real-time monitoring of theassociation and dissociation of the interacting molecules. Thesensorgram provides quantitative information in real-time on specificityof binding, active concentration of molecule in a sample, kinetics andaffinity.

KINEXA. The KINEXA technology (Sapidyne Instruments, Boise, Id.)measures binding constants to characterize biomolecular binding eventsin the solution phase, not binding events between a solution phase and asolid phase. In solution, the binding partners reach equilibrium aftersufficient incubation. The unbound molecules are quantified with atitration, which reflects the portion of molecules bound to thepartners. The KINEXA method does not require modification of moleculesunder study. With KINEXA, the reaction being measured occurs betweenunmodified molecules in solution. Therefore, concerns of howmodification alters “native” binding reactions are eliminated. TheKINEXA method allows a wider range of binding constants as tight as10⁻¹³ M. The KINEXA software performs data analyses, which are based onexact solutions to classic binding equations (K^(d) mathematics), notpseudo first-order approximations. KINEXA does not require arbitrarydata manipulations or range selections.

As shown in Table 8, antibody 19.3 had a 4.8 nM affinity for ADDLs ascompared to a 150 nM affinity for monomer Aβ in the BIACORE assay. Thethirty-fold selectivity of antibody 19.3 for ADDLs over Aβ monomer wasmarkedly better than that seen for the parental antibody, h3B3, whichexhibited only a 10-fold preference for ADDLs versus Aβ monomer.

TABLE 8 Ratio (Aβ Antibody ADDLs (nM) Aβ1-40 (nM) monomer/ADDL) 3B3 10.0104.6 ~10 19.3 4.8 150.0 ~31

Similarly, antibody 19.3 was evaluated in a KINEXA-based equilibriumconstant measurement. As shown in Table 9, antibody 19.3 had anequilibrium constant of 2.7 nM, which represents more than a six-foldpreference for ADDL oligomers versus Aβ40 monomer binding in the sameassay.

TABLE 9 Ratio (Aβ Antibody ADDLs (nM) Aβ1-40 (nM) monomer/ADDL) 3B3 3.345.0 ~13.6 19.3 2.7 16.7 ~6.2

EC₅₀ of 19.3 for Aβ Oligomers and Aβ1-40 in One-Sided ELISA Assay. EC₅₀represents the half-maximal total Aβ oligomer binding. High proteinbinding plates were coated at either 100 pmol/well Aβ1-40 or 50pmol/well Aβ oligomers in PBS, overnight at 4° C. Next day, plates werewashed five times with PBS+0.05% TWEEN 20 and blocked overnight withcasein blocking buffer (Thermo Scientific, Waltham, Mass.) and 0.05%TWEEN 20. The 19.3 antibody was tested at 0 to 15 μg/ml in a 12-pointthree-fold dilution series. After two hours at room temperatureincubation, the plates were washed and alkaline phosphatase-conjugatedanti-human IgG (ThermoScientific, Waltham, Mass.) was added at 0.08μg/ml. After incubation for 45 minutes at room temperature, the plateswere washed and TROPIX CDP star (Applied Biosystems, Foster City,Calif.) was added. Luminescence was detected after minutes on anENVISION plate reader (PerkinElmer, Waltham, Mass.). Curve fits werecompleted using GraphPad Prism (GraphPad Software, Inc., San Diego,Calif.) software. This analysis indicated that the 19.3 antibody (IgG2isotype) has an EC₅₀ of approximately 1.6 nM and 4.3 nM for Aβ oligomersand Aβ1-40 monomer, respectively, in the one-sided ELISA assay (FIG.5A). In this format the 19.3 antibody demonstrated approximatelythree-fold greater maximum binding for Aβ oligomers as compared to Aβ40monomer, while the potency was approximately 3.7-fold greater.

Competitive Binding Assays with Aβ Oligomers and Aβ Monomer. To moreaccurately represent an in vivo CSF sample, where both Aβ oligomers andAβ monomers would be present, the affinity of 19.3 for Aβ oligomers inthe presence of Aβ1-40 monomer was tested in a competitive ELISA format.

The ELISA plate was prepared by first coating with a preparation of Aβoligomers at 50 pmol per well and then adding the 19.3 antibody at afinal concentration of 2 nM to each well. This concentration of 19.3,i.e., 2 nM, represents the EC₅₀ concentration for Aβ oligomers bindingdetermined in the one-sided ELISA (FIG. 5A). Adding Aβ1-monomer in atitration curve to competitively remove 19.3 from the Aβ oligomer-coatedsurface resulted in an EC50 of 5.5 μM. Aβ1-40 monomer-coated plates wereprepared in the same way, using 100 pmol/well. The 19.3 antibody wasapplied at 4 nM to each well in the casein blocking buffer matrix andallowed to interact with Aβ oligomers or Aβ1-40 for 30 minutes at roomtemperature with shaking. A 12-point, three-fold concentration curvestarting at 10 μM, for either Aβ oligomers or Aβ1-40, was applied to theantibody containing wells. For plates coated with Aβ oligomers, Aβ1-40was added to the wells; for Aβ1-40 plates, Aβ oligomers were added tothe wells. The plates were incubated for one and half hours at roomtemperature. Both detection of residual antibody binding and the EC₅₀calculations were determined as in the one-sided ELISA assay.

This analysis indicated that adding Aβ1-40 monomer in a titration curveto competitively remove 19.3 from Aβ oligomer-coated surface resulted inan EC₅₀ of 5.5 μM (FIG. 5B). When 100 pmol per well of Aβ1-40 monomerwas used to coat the ELISA plate and Aβ oligomers were used to competefor antibody binding, the EC₅₀ was 8.7 nM. This indicated that 19.3 hadan affinity for Aβ1-42 oligomers compared to Aβ1-40 monomers of ˜630:1in a competitive binding assay. Alternatively stated, the concentrationof Aβ1-40 required to displace 50% of 19.3 from Aβ oligomers wasapproximately 600-fold higher than the concentration of Aβ oligomersrequired to displace 19.3 binding to Aβ1-40. Concentrations up to 0.2 pMof Aβ oligomers have been reported in CSF from AD patients(Georganopoulou, et al. (2005) Proc. Natl. Acad. Sci. USA 102:2273-2276)as compared to 1500 pM of Aβ monomer. Thus, the sensitivity andselectivity of 19.3 for Aβ oligomers indicated a potential utility in asandwich ELISA to detect Aβ oligomers above background levels of Aβmonomer.

ALPHALISA Assay. The ALPHALISA technology (PerkinElmer) is a bead-basedimmunoassay designed for the detection of analytes in biologicalsamples. This chemiluminescent assay exhibits remarkable sensitivity,wide dynamic range and robust performance that compares advantageouslywith conventional ELISA. The selectivity and sensitivity the 19.3antibody for ADDLs versus monomeric Aβ (Aβ1-40) in the ALPHALISA assaywas determined. This analysis indicated that a signal at 0.2 pM of ADDLswas greater than a signal at 1000 pM of Aβ1-40, indicating an ADDLversus monomeric Aβ selectivity of approximately 5000 in this assay.

Immunohistochemistry. Immunohistochemical analysis of tissues fromTg2576 mice indicated that the 19.3 antibody did not bind vascularplaques, exhibited essentially no binding to dense core plaques and noplaque clearance in Tg2576 mice.

Example 8 Biophysical Characterization of Anti-ADDL Antibody 19.3

Biophysical characterization to assess the potential for antibodyaggregate formation was carried out to show that the anti-ADDLantibodies were stable under various conditions. Anti-ADDL antibody 19.3was concentrated to >50 mg/mL and placed in a number of formulationswith a pH ranging from 5.0 to 8.0. Two sets of samples were incubated at37° C. and 45° C. for one week. A third set of samples was placed at−70° C. to initiate a series of five freeze/thaw cycles. Size exclusionchromatography analysis indicated that the antibody preparations werepredominantly (>95%) in the monomer state, with a small amount ofdimers, which is typical for monoclonal antibody preparations. Theamount of dimers and higher molecular weight oligomers did not increaseafter the temperature stress across all buffers and no fragmentation wasobserved. As summarized in Table 10, the near ultraviolet turbidityanalysis also indicated lack of aggregation. The freeze/thaw stressedsamples showed buffer-dependent increase in turbidity, which wascomparable to other monoclonal antibodies. Viscosity at 50 mg/mL wasbelow 2 centipoise. Differential scanning calorimetry also revealedacceptable thermal stability, with Fab unfolding at about 72° C. and theleast stable CH2 domain unfolding above 65° C. Taken together, antibody19.3 demonstrated very good structural stability.

TABLE 10 Antibody Initial Aggregates (%) Initial Fragments (%) 19.3 2.20.0 Control 1 1.6 0.4 Control 2 2.6 0.0

Example 9 Preparation of 19.3 Variants

An assessment of the amino acid sequence of the 19.3 antibody wasconducted to identify potential sites of deamidation. Asparagine andaspartic acid residues present in the CDRs of therapeutic antibodies canundergo deamidation and isoaspartate formation (Valsak & Ionescu (2008)Curr. Pharm. Biotech. 9:468-481; Aswad, et al. (2000) J. Pharm. Biomed.Anal. 21:1129-1136), the formation of which can alter the bindingpotency of an antibody and, in turn, reduce antibody effectiveness foruse as a therapeutic. Therefore, the asparagine residue at position 33of the light chain CDR1 of antibody 19.3 was altered. Variants of the19.3 antibody were produced (Table 11) with the substitution of serine,threonine or glutamic acid for the asparagine at position 33 in CDR1.The substitution of aspartic acid for the asparagine as position 33 wasalso generated as a control.

The mutagenesis of the asparagine at position 33 (N33) of the lightchain CDR1 for the antibody 19.3 into N33S, N33T, N33E, or N33D wascarried out by site-directed mutagenesis from the wild-type expressionvector of pVl JASN-GS-19.3-LCK using QUIKCHANGE IT XL Site-DirectedMutagenesis Kit (Agilent Technologies, La Jolla, Calif.). The codon AATfor N was mutated to AGT for S in 19.3 N33S, ACT for T in 19.3 N33T, GAAfor E in 19.3 N33E, or GAT for D in 19.3 N33D. Additional mutations atthe asparagine at position 35 (N35) of CDR1 were also generated andcombined with the N33S mutation (Table 11). Furthermore, mutations atthe asparagine at position 58 in the CDR2 of antibody 19.3 were prepared(Table 12). All new codons in were confirmed by DNA sequence analysis.To generate full-length IgG antibodies for these variants, therespective light chain plasmids were paired with the cognate heavy chainplasmid, pVlJNSA-19.3-HCG2, for transient transfection in 293 FREESTYLEcells (Invitrogen, Carlsbad, Calif.). The expression and purificationmethods were described above.

Table 11 summarizes the amino acid sequence of CDR1 of the light chainof the variants compared to the CDR1 of the light chain for the parentalantibody, 19.3. The present invention provides the variants of 19.3whose light chain CDR1 is as set out in Table 11 below and whose CDR2and CDR3 light chains and all heavy chains are as set for 19.3 itself.

TABLE 11 Antibody LC-CDR1 Sequence SEQ ID NO: 19.3 (parental)RSSQSIVHSNGNTYLE 14 19.3 N33S RSSQSIVHSSGNTYLE 46 19.3 N33TRSSQSIVHSTGNTYLE 47 19.3 N33A RSSQSIVHSAGNTYLE 48 19.3 N33ERSSQSIVHSNGNTYLE 49 19.3 N33D RSSQSIVHSDGNTYLE 50 19.3 N33S-N35QRSSQSIVHSSGQTYLE 51 19.3 N33S-N35S RSSQSIVHSSGSTYLE 52 19.3 N33S-N35TRSSQSIVHSSGTTYLE 53 19.3 N33S-N35A RSSQSIVHSSGATYLE 54

Table 12 summarizes the amino acid sequence of CDR2 of the light chainof the variants compared to the CDR2 of the light chain for the parentalantibody, 19.3. The present invention provides the variants of 19.3whose light chain CDR2 is as set out in Table 12 below and whose CDR1and CDR3 light chains and all heavy chains are as set for 19.3 itself.

TABLE 12 Antibody LC-CDR2 Sequence SEQ ID NO: 19.3 (parental) KASNRFS 1519.3 N58Q KASQRFS 55 19.3 N58S KASSRFS 56 19.3 N58T KASTRFS 57 19.3 N58AKASNRFS 58

The 19.3 variants were subsequently evaluated to determine whether themutations had any effect on the stability of the antibody. Aliquots ofpurified variant antibodies, along with the 19.3 parental antibody, wereincubated under various conditions at 4° C., 25° C. or 40° C. for amonth before being subjected to ELISA analysis. High protein bindingplates (Costar, Corning, Lowell, Mass.), were coated with target ligandin PBS overnight at 4° C. The concentration of coating protein was 50pmol/well for ADDLs. ADDLs were generated as described in Example 1. Onethe next day, plates were washed five times with PBS I 0.05% TWEEN 20(Sigma Aldrich, St. Louis, Mo.) and blocked overnight with caseinblocking buffer (Thermo Scientific, Waltham, Mass.) and 0.05% TWEEN 20.Three representative antibodies, 19.3, 19.3 N33S, and 19.3 N33T weretested at 15 μg ml to 0 μg/ml in a 12-point three-fold dilution series.After 2 hours at room temperature incubation, the plates were washed andalkaline phosphatase-conjugated anti-human IgG (ThermoScientific,Waltham, Mass.) was added at 0.08 μg/ml. After 45 minutes at roomtemperature incubation, the plates were washed and TROPIX CDP-Starchemiluminescent substrate (LIFE TECHNOLOGIES, Carlsbad, Calif.) wasadded. Luminescence was detected after 30 minutes on an ENVISIONmicroplate reader (PerkinElmer, Waltham, Mass.). Curve fits werecompleted using GRAPHPAD PRISM software (GraphPad Software, Inc., SanDiego, Calif.).

As shown in FIGS. 6B and 6C, antibodies 19.3 N33S and 19.3 N33T hadenhanced binding stability compared to the 19.3 parent (WT, FIG. 6A)following a one-month incubation at varying temperatures. A summary ofthe EC₅₀s of these antibodies at the various incubation temperatures isprovided in Table 13.

TABLE 13 Antibody EC₅₀ (nM) Antigen Incubation 19.3 19.3 N33T 19.3 N33SbADDL 0 timepoint 1.1 15.5 7.8  4°, 1 month 1.7 11.6 8.6 25°, 1 month2.1 15.7 12.8 40°, 1 month 5.9 23.5 10.1 Aβ1-40 0 timepoint 10.1 332.155.1  4°, 1 month 16.3 306.8 59.1 25°, 1 month 22.1 ND 24.3 40°, 1 month88.8 96.3 29.9

EC₅₀s of several of the 19.3 variants were determined and it was foundthat the variants maintained specificity for ADDLs in an ELISA assay(Table 14)

TABLE 14 EC₅₀ (nM) Antibody ADDL Aβ1-42 19.3 0.8 18 19.3 N33S 1.7 15019.3 N33T 3.1 244 19.3 N33D 0.82 28 All antibodies were IgG2.

Example 10 Aβ Oligomer Preferring Antibodies in Aβ Oligomer-SelectiveSandwich ELISA

In a screen of capture and detecting antibody pairs in a sandwich ELISAformat, the combination of 19.3 as the capture antibody with either7305, an anti-Aβ oligomer antibody (20C2, U.S. Pat. No. 7,780,963, whichis incorporated herein by reference in its entirety) or 82E1(Immunobiological Laboratories (IBL), Inc., Minneapolis, Minn.)performed comparably in casein blocking buffer in an Aβ oligomerstandard curve, each giving a limit of detection (LeD) under 4 pg/mL(FIG. 7A). Use of an anti-Aβ monomer antibody as both capture anddetection antibody has been reported as an Aβ oligomer assay, however,absolute levels of sensitivity or selectivity were either not reported(6E10/6E10; Gandy, et al. (2010) Ann. Neurol. 68:220-230), orselectivity was below that desired for an assay to measure Aβ oligomersin human CSF (82E1/82E1; Xia, et al. (2009) Arch. Neurol. 66:190-199).

To determine the sensitivity of 6E10 and 82E1, these antibodies wereused in sandwich ELISA assays. In this analysis, identical antibodieswere used for both capture and detection antibodies, e.g., 6E10/6E10(FIG. 7B) and 19.3/19.3 (FIG. 7C), as well as sandwich ELISA assay pairsusing 19.3 as a capture antibody only (FIG. 7A, with 82E1 detection).This analysis indicated that 6E10/6E10 and 19.3/19.3 both demonstratedapproximately one hundred fold reduced sensitivity compared to either19.3/7305 or 19.3/82E1.

The 19.3/82E1 ELISA utilizing luminescence detection technology(ENVISION Multilabel plate reader, PerkinElmer, Waltham, Mass.) (FIG.7A), generated a LoD of approximately 1.3 pg/mL. In this assay format,the lower limit of reliable quantification (LLoRQ) of Aβ oligomer was4.2 pg/mL (with coefficients of variance less than 20% at this lowestmeasure) and the assay was approximately 1000 fold-selective for Aβoligomer signal as compared to Aβ40 monomers. While this assay was usedto evaluate Aβ oligomer preparations, it may not be sensitive enough toreliably detect Aβ oligomer levels in human CSF at levels suggested byprevious estimates (Georganopoulou, et al. (2005) Proc. Natl. Acad. Sci.USA 102:2273-2276).

Example 11 Aβ Oligomer-Selective Sandwich ELISA with ImprovedSensitivity

Both the 19.3 and 7305 (19.3×7305) and the 19.3 and 82E1 (19.3×82E1)antibody pairs were evaluated in a sandwich ELISA using a paramagneticmicroparticle detection immunoassay system, ERENNA Immunoassay System(SINGULEX, Almeda, Calif.) to determine if assay sensitivity could beimproved further for the measurement of Aβ oligomers in human andnon-human primate fluid samples.

Capture Antibody Labeling. Binding of the capture antibody to DYNABEADS(microparticle (MP) beads) was achieved by removing supernatent from 50μl of DYNABEADS using a magnet. The DYNABEADS were resuspending in 200μl of an antibody binding and washing buffer, e.g., RIPA buffer (CellSignaling Technologies, Beverly, Mass.), containing 5 μg of the captureantibody. The mixture was incubated for 10 minutes with rotation at roomtemperature. The supernatent was removed from capture antibody/MP beadcomplex with a magnet. The complex was washed with 200 μl of the bindingand washing buffer.

Coupling of Capture Antibody to DYNABEADS (MP beads). The captureantibody-coupled MP beads (5 μg 19.3/50 μl MP bead complex) were washedtwice in 200 μL of the conjugation buffer (20 mM sodium phosphate, 0.15M NaCl (pH 7-9)), placed on a magnet and the supernatant was discarded.The capture antibody/MP beads were resuspended in 250 μl 5 mM BS3solution (Bis(sulfosuccinimidyl) suberate in conjugation buffer). Theresuspended beads were incubated at room temperature for 30 minutes withtilting/rotation. The cross-linking reaction was quenced by adding 12.5μl of a quenching buffer (1M Tris-HCl, pH 7.5) and subsequentlyincubated at room temperature for 15 minutes with tilting/rotation. Thecross-linked MP beads were washed three times with 200 μl PBS-T. The MPbeads were diluted to 100 μg/mL in assay buffer for use in the assayprotocol.

Detection Antibody Labeling. ALEXA FLUOR 546 (Invitrogen, Carlsbad,Calif.) was coupled to the detection antibody according to themanufacturer's protocol. Briefly, detection antibody was diluted to 1mg/mL and one-tenth volume of 1M sodium bicarbonate buffer was added.This solution of detection antibody (100 μL) was added to the vial ofALEXA FLUOR 546 dye, and the vial was capped, gently inverted todissolve the dye and stirred at room temperature for 1 hour. The columnswere spun to separate any unlabeled fluorescent tag from the detectionantibody and the antibody was loaded onto a Component C (BIOGEL P-30,BioRad, Hercules, Calif.) fine size exclusion purification resin. Afterthe gel buffer drained away, 100 μL detection antibody and dye reactionvolume were added onto the center of the resin at the top of the spincolumn and absorbed into the gel bed. To the column was slowly added, atroom temperature, 100 μL of an elution buffer (0.01 M potassiumphosphate, 0.15 M NaCl, pH 7.2, with 0.2 mM sodium azide). Additionalelution buffer was added and as the column ran, the column wasilluminated to visualize the front of the dyed/tagged antibody. Thefirst column dye line was the labeled antibody. Free dye remained in thecolumn bed and was discarded with the spin column.

The Aβ oligomer sandwich ELISA was carried out using a paramagneticmicroparticle-based immunoassay platform (ERENNA immunoassay system,SINGULEX, Almeda, Calif.) to determine oligomer levels in CSF samples orAβ oligomer standards. Microparticles (MPs) for capture were prepared bybinding 12.5 μg of the capture antibody per mg of MPs. The captureantibody-bound MPs were diluted to 100 pg/mL in assay buffer (Trisbuffer with 1% TRITON X-100, d-desthiobiotin, 0.1% bovine serum albumin)and added at 100 μL to 100 μL of CSF sample or standards (diluted inTris buffer and 3% bovine serum albumin), followed by incubation for twohours at 25° C. The MPs were retained via a magnetic bed, and unboundmaterial was removed in a single wash step using assay diluent using theTHYDROFLEX plate washer (Tecan, Männedorf, Switzerland). The ALEXAFLUOR-labeled detection antibody was diluted to a final concentration of500 pg/mL and filtered through a 0.2 μm filter (Pall 4187, FortWashington, N.Y.). The detection antibody was added to 20 μL/well ofindividual sample particles. The ELISA plates were incubated for onehour at 25° C., while shaking in a Jitterbug (Boekel, Feasterville,Pa.). The wells were washed four times with assay buffer to remove anyunbound detection reagent. MP/capture antibody/A11 oligomer/detectionantibody complexes were transferred to a new plate, buffer was aspiratedoff and 10 μL/well of elution buffer was added, followed by a 5 minuteincubation at 25° C., while shaking in a Jitterbug at speed 5. Eluted,fluor-labeled detecting antibody was transferred to a 384 platecontaining 10 μL/well neutralization buffer and read on a paramagneticmicroparticle detector (ERENNA, SINGULEX, Alameda, Calif.) at 60 secondsper well read time.

While paramagnetic microparticle immunoassays, such as the ERENNAImmunoassay System, have been used for biomarkers present in abiological sample in the nanomolar (nM) range, as observed for Aβ1-40and Aβ1-42, it has not been previously demonstrated for as animmunoassay system that can specifically and reliably detect a biomarkerpresent in a CSF sample in the femtomolar (fM) range, such as with Aβoligomers. Without wishing to be bound by any theory, it is believed,and has demonstrated, that the specificity and sensitivity of the assaysherein are attributable to the specificity and sensitivity of theanti-ADDL antibody pair selected and used in the sandwich ELISA.Similarly, while the ERENNA Immunoassay System is used herein toillustrate the claimed assay, it is possible that other detectionsystems having comparable sensitivities could be employed in theinventive methods.

The 19.3×7305 sandwich ELISA was conducted using the ERENNA ImmunoassaySystem (SINGULEX, Almeda, Calif.), covalently-coupling the 19.3 antibodyto the ERENNA microparticle (MP) beads (hereinafter “19.3/MP beads”).The 19.3/MP beads were then mixed with buffer containing a standardcurve of either Aβ oligomer or monomeric Aβ40. The resulting 19.3/MPbead/Aβ oligomer or Aβ40 complex (hereinafter “Aβ oligomer complex”) waswashed and either a fluorescently-tagged 7305 or 82E1 detection antibodywas bound to the Aβoligomer complex. The ERENNA instrument, using aproprietary detection technology capable of single-molecule counting(see U.S. Pat. No. 7,572,640), measured the fluorescently-labeleddetection antibody following its release from the sandwich ELISA. Asshown in Table 15, data from the 19.3×7305 assay, using a two-folddilution of the Aβ oligomer standard in buffer, aligned with a lineartwo-fold dilution of fluorescent signal (detected events mean). Signalsgenerated by neat rhesus CSF, or CSF to which a standard curve of Aβoligomers was introduced, demonstrated that the fluorescent signalattributed to binding of the tagged 7305 antibody was equivalent in bothcases, while the 19.3×82E1 sandwich assay was able to detect spiked Aβoligomers across the full standard curve. In the assay format using 7305as the detection antibody, this was indicative that there was anon-specific background (from something present in the rhesus CSF)saturating over the range of the Aβ oligomers dilution series that wassufficient to detect Aβ oligomers in buffer alone. Subsequently, thefluorescent signal was found to be identical to that for a nakedmicroparticle, even in the absence of the 19.3 antibody coupling, whichwas also consistent with a non-specific signal due to 7305 antibodycross-reactivity.

TABLE 15 Expected Interp Standard [ADDLs] DE [ADDLs] % Diluent pM MeanSD CV % pM Mean SD CV % Recovery Standards 5.00 5579 506 9 5.1 0.5 10103 Diluent 1.67 1942 235 12 1.7 0.2 13 100 0.56 691 152 22 0.5 0.1 2596 0.19 324 43 13 0.2 0.1 17 116 0.06 131 34 26 0.1 0.1 49 88 0.00 72 2839 ND Rhesus 5.00 9097 88 1 CSF- 1.67 9112 195 2 Depleted 0.56 8721 1662 0.19 8785 269 3 0.06 8744 273 3 0.00 8678 519 6 Rhesus 5.00 10353 2372 CSF-Non- 1.67 9719 495 5 Depleted 0.56 9902 546 6 0.19 9971 319 3 0.069721 329 3 0.00 10515 282 3 n = 3 for each experiment.

As an alternative, the 7305 detection antibody was replaced with 82E1,also coupled to a fluorescent tag, in the Aβ oligomer-selective sandwichELISA (FIG. 9A) developed using the ERENNA Immunoassay System. Like19.3, the 82E1 antibody has reported in ELISA formats to detect Aβoligomers in AD brain (Xia, et al. (2009) Arch. Neurol. 66:190-199). Asshown in Table 16, this assay eliminated the non-specific signal in boththe neat and Aβ oligomer-depleted rhesus CSF, further suggesting thatthe 7305 antibody had been the source of the non-specific signal.Without wishing to be bound by any theory, the high background signalobserved for the 19.3/7305 antibody pair was believed to be due to CSFfibrinogen binding to the MP beads, which was not observed for the19.3/82E1 antibody pair. This alternative assay generated a LoD of theAβ oligomer standards at 0.04 pg/mL, a LLoRQ at 0.42 pg/mL and 5000-foldselectivity of the assay for Aβ oligomers over Aβ 40 monomer (FIG. 8).On the basis of these findings, this assay format was selected forfurther characterization.

TABLE 16 19.3/7305 19.3/82E1 Parameter Antibody Pair Antibody Pair Slopedetected events (pM) 1,200 4,000 Background 70 100 LoD (pM) 0.01 0.01LLoRQ (pM) 0.16-0.49 0.12 Aβ40 monomer Cross Reactivity 0.02% 0.04%Depleted Rhesus CSF (pM) 80 <0.12 Non-Depleted Rhesus CSF (pM) 200 0.35

Example 12 Pharmacodynamic (PD) Assay

Using the findings above, a selective Aβ oligomer sandwich ELISA wasdeveloped, using the 19.3 and 82E1 antibody pair, to detect and measurethe levels of Aβoligomers in a CSF sample. This assay will heretofore becalled the pharmacodynamic (PD) assay for its use to assess changes inthe analyte, i.e., Aβ oligomer, levels following treatment to inhibitproduction, increase clearance, or otherwise modify Aβ oligomer levels(FIG. 95). The PD assay can also be used to differentiate AD from non-ADpatients, i.e., diagnostic, to monitor the progression of the disease,i.e., prognostic, or to monitor the therapeutic potential of adisease-modifying treatment to change Aβ oligomer concentrations.

The PD assay, as described in the previous Example with reference toFIG. 9B, placed the 19.3 antibody coupled to a paramagneticmicro-particle (MP) bead (MP bead/19.3) into a well on an ELISA plate.To the well was added either a human CSF or an Aβ oligomer standard (ina dilution series added to a Tris buffer and bovine serum albumin). AnyAβ oligomer present in the well was bound by the 19.3/MP bead and theexcess solution was washed away. Fluorescent-labeled 82E1, as thedetection antibody, within an assay buffer (Tris buffer with 1% TRITONX-100, d-desthiobiotin, BSA), was added to the washed MP bead/19.3/Aβoligomer complex and incubated, to bind the Aβ oligomer complex. Theresulting MP bead/19.3/Aβ oligomer/82E1 complex was washed with anelution buffer and the fluorescent-labeled 82E1 antibody was eluted withany unbound antibody. Detection with the paramagnetic micro-particledetector, such as the ERENNA instrument, in which the solution flows byand is excited by a laser, allows the detection of single molecules(fluorescent tag emits photons of a specific light wavelength) togenerate and measure a fluorescent signal, equivalent to the moleculesdetected, i.e., Aβ oligomer. A standard curve of Aβ oligomers, asmeasured with the ERENNA instrument, as compared to Aβ monomers is shownin FIG. 8.

Example 13 Aβ Oligomers in Human CSF

The 19.3×82E1 Aβ oligomer-selective sandwich ELISA of the previousExample was used to measure endogenous levels of Aβ oligomers in humanCSF samples (FIGS. 10A and 10B). In two separate sample cohorts, thefluorescent signal, generated by the presence of Aβ oligomers, wassignificantly elevated in AD (clinically diagnosed using a MMSE scorebelow 25 as probable AD) CSF as compared to either young or healthy agematched controls. The absolute levels of Aβ oligomers observed were2.1±0.61 pg/mL in AD (n=20) and 0.53±0.26 pg/mL in age-matched control(n=10) in CSF samples from Precision Medicine (Solana Beach, Calif.)with a t-test, two way Mann-Whitney score of p<0.0004 (FIG. 10A). Theabsolute levels of A3 oligomers observed were 1.66±0.5 pg/mL in AD(n=10) and 0.24±0.05 pg/mL in control (n=10) in CSF samples fromBioreclamation (Hicksville, N.Y.), with a t-test, two way Mann-Whitneyscore of p<0.0021 (FIG. 10B). Combining the two cohorts, 90% of thediagnosed AD CSF samples were above the LLoRQ of 0.42 pg/mL, while only20% of the age-matched control or 10% of the young controls were abovethis limit. All values were above the LoD of 0.04 pg/mL. Aβ40 and Aβ42monomer levels were measured in the CSF samples obtained fromBioreclamation (FIGS. 11A and 11B, respectively) and were comparablebetween the AD and control CSF for Aβ1-40 (FIG. 11A), while they weresignificantly reduced in the AD samples for Aβ1-42 (FIG. 11B). This hasbeen previously reported as a feature of AD CSF (De Meyer, et al. (2010)Arch. Neurol. 67:949-956; Jack, et al. (2010) Lancet Neural. 9:119-128)and confirmed the correct diagnosis of these samples. Without wishing tobe bound to any theory, it is believed that the lower levels of Aβ1-42in the AD CSF samples is due to retention of Aβ1-42 in the amyloiddeposits of the AD brain. The ability to specifically detect andquantify these observed differences suggests that these biomarkers canbe used as a diagnostic and prognostic measure for AD.

For a diagnostic assay, the signal, i.e., the level of Aβ oligomers,detected from the assay herein would typically be greater thanthree-fold higher for an AD patient (to a level >0.5 pg/mL) as comparedto the signal observed for non-AD patients. This is consistent with thedata, shown in both FIG. 10A, in which the levels of Aβ oligomers in theAD CSF compared to age-matched controls were four-fold higher, and inFIG. 10B, wherein levels of Aβ oligomers in AD CSF was eight-foldhigher. This data also support the use of the Aβ oligomer assay hereinto identify patients at early stages of disease (i.e., a prognosticassay). Age is the biggest risk factor for the development of AD and thedifferences observed between AD and age-matched controls were smallerthan between AD and young controls. Similarly, for a prognostic Aβoligomer assay, patients having a MMSE of below 25 would have a detectedAβ oligomer signal of >0.5 pg/ml, (four- to eight-fold higher thanpatients with MMSE above 25/normal) as compared to the signal detectedfor A31-42 monomer, which is approximately two-fold lower in the AD CSFcompared to controls. Using an MMSE score of 25 as a cutoff (Mungas(1991) Geriatrics 46(7): 4-58), wherein an MMSE score above 25 isconsidered “normal healthy” and below is considered as either mildlycognitively impaired, or as having AD, it would be expected that an Aβoligomer level of ≧0.5 pg/mL is indicative of a patient with an MMSEscore below 25 (FIG. 12).

Example 14 Target Engagement (TE) Assay

Using the findings above, a TE assay was developed to measure Aβoligomers bound in vivo to a therapeutic (capture) antibody. As such,the TE assay can be used to measure levels of Aβ oligomers bound to atherapeutic antibody to confirm engagement of the Aβ oligomer by thetherapeutic. Without wishing to be bound by any theory, it is believedthat the level of Aβ oligomers bound to a therapeutic anti-Aβ oligomerantibody will be lower in CSF samples from subjects who have beentreated over time with said therapeutic. Levels of bound Aβ oligomersthat increase or are unchanged post-administration would suggest thatthe therapeutic is not suitable for the treatment of AD. Alternatively,it may be the case that merely by sequestering the Aβ oligomers andbinding them to the therapeutic antibody, a benefit may be obtained inacute performance, due to reduced interaction with neurons in the brain.However, this benefit may not be associated with a change in Aβoligomers per se. The target engagement assay would assess, at aminimum, the ability of a therapeutic antibody to engage Aβ oligomerswithin the CSF.

In this assay an anti-human IgG2 antibody×82E1 antibody pair is used todetect and quantify levels of bound Aβ oligomers in a CSF sample from apatient treated with the anti-Aβ oligomer 19.3 (IgG2) antibody, i.e., atherapeutic antibody (FIG. 9B). To demonstrate the ability of Aβoligomer-specific antibodies to engage Aβoligomers in a human CSFmatrix, 19.3/Aβ oligomers complexes were generated within human CSF byspiking the CSF with the anti-Aβ oligomer antibody 19.3 to levelsbelieved to be present at 24 hours in rhesus monkey dosed IV with 20 m/k(100 ng/mL, FIG. 13). To this 19.3-spiked human CSF sample was added anescalating amount of Aβ oligomer standards, both matching endogenous Aβoligomer concentrations (0.1-5.0 pg/mL) (FIGS. 10A and 10B) and alsoraising them significantly above normal ranges. The 19.3×Aβ oligomercomplexes formed in human CSF were captured onto 96-well ELISA platescoated with either antibody to human kappa light chain or antibody tohuman IgG2 (both from Southern Biotech, Birmingnam, Ala.) at 0.5 μg perwell in a sodium bicarbonate buffer overnight at 4° C. (BupHCarbonate-Bicarbonate Buffer pack, Thermo Fisher Scientific Inc, WalthamMass.). Next day, the wells were washed with PBS-T and blocked overnightat 4° C. with 200 μL/well casein buffer in PBS with 0.1% TWEEN 20 added.The 19.3 antibody was spiked into a casein buffer (Thermo FisherScientific Inc, Waltham Mass.) or human CSF in microcentrifuge tubes(Axygen, Inc., Union City, Calif.). The Aβ oligomers were spiked atvarying concentrations to give a standard curve, keeping the 19.3 levelsconstant. The samples were agitated at 4° C. for one hour to enableformation of the antibody (19.3)/Aβ oligomer complexes. One hundred μlsample/well was applied to either an anti-human IgG2 or an anti-humankappa-coated plate (n=3) and incubated overnight at 4° C. on a plateshaker. Next day, the plates were washed five times with PBS-T andBiotin-82E1 (IBL, Minneapolis, Minn.) was added at 100 μl/well, diluted1:5000 in casein blocking buffer (Sigma-Aldrich Co., St. Louis, Mo.),0.1% TWEEN 20 for one hour at room temperature. The plates were washedagain with PBS-T, and Neutravidin-AP (ThermoFisher, Waltham, Mass.) wasdiluted 1:20000 in casein buffer, then added for 30 minutes at roomtemperature. Additional PBS-T washes were followed with TROPIX CDP starluminescence substrate (Applied Biosystems, Foster City, Calif.) appliedfor 30 minutes. Luminescence was quantified on an ENVISION plate reader(PerkinElmer, Waltham, Mass.).

The anti-Aβ oligomer antibody 19.3 was sufficiently recognized by bothanti-human kappa and anti-human IgG2 in buffer (FIGS. 14A and 14B,filled triangles), as the antibody contains both of these features. Asshown in FIG. 14A (filled circle, CSF), the assay using anti-human IgG2as the capture antibody and 82E1 as the detection antibody, to detectand measure the 19.3/Aβ oligomer complex, resulted in significantlybetter sensitivity in human CSF as compared to the assay usinganti-human kappa as the capture antibody (filled circle, CSF, FIG. 14B).Both assays had equivalent sensitivity in casein buffer. Use ofanti-human kappa to capture the 19.3/Aβ oligomer complex resulted inless sensitivity, to a LoD of 42 pg/mL Aβ oligomer bound to 100 ng/mL19.3, perhaps due to higher background levels of IgG species with akappa light chain in human CSF as compared to IgG2 species, whichresulted in greater sensitivity for the assay format using an anti-IgG2.Following dosing of either human or experimental animals with 19.3 or arelated IgG2 anti-Aβ oligomer antibody as a therapeutic antibody, onewould expect the therapeutic antibody to be represented in the CSF at0.1-0.20 of the dosed level (Thompson (2005) Proteins of theCerebrospinal Fluid, Elsevier Academic Press, New York, N.Y.). Thetherapeutic antibody present in the CSF would be bound to available Aβoligomers, the 19.3 (IgG2)/Aβ oligomer complexes would be captured withthe anti-IgG2 capture antibody through the anti-human 19.3, IgG2,antibody, and the Aβ oligomer complexes would then be detected with82E1. The sensitivity of this platform would likely improve using aparamagnetic microparticle detection system, such as the ERENNAimmunoassay system (SINGULEX, Alameda, Calif.), utilized in the PD assayherein.

Over time, following therapeutic treatment with an anti-Aβ oligomerantibody, it is expected that the signal detected for the19.3/Aβ-oligomer complexes would be reduced (as compared topre-treatment levels). The amount of bound Aβ oligomer, whether asmeasured for these complexes acutely or after a period of therapeutictreatment, represents the proportion of the therapeutic antibody engagedwith the target, i.e., Aβ oligomers, and could serve as a surrogate forthe efficacy of the therapeutic antibody.

Example 15 Additional Antibody Characterization

A solution-based binding assay was used to determine the specificity andaffinity of anti-ADDL antibodies to different amyloid beta peptidepreparations (ADDL, fibril, Aβ-1-40, Aβ1-20). A quantitative ELISA wasused that was capable of capturing the linear range of dose-response ofmonoclonal antibodies against ADDLs coated on NUNC plates. Based on thisinformation, a fixed concentration of monoclonal antibody was selectedthat could give consistent OD signals in ELISA just above assay noise(OD450 nm reading around 0.2 to 0.5). Anti-ADDL antibody at this fixedconcentration was then incubated with different amyloid beta peptidesubstrates (ADDL, fibril, Aβ1-40, Aβ1-20) in 20 point titrations insolution at room temperature overnight to reach equilibrium. Thequantity of free anti-ADDL antibody within the mixture was determinedthe next day in a quantitative ELISA with one hour incubation on regularELISA plates. The fraction of bound anti-ADDL antibody was calculatedand the correlations of bound anti-ADDL antibody to titration of freeligand (substrates) were used to derive K_(D), using the GRAFIT program(Erithacus Software, Surrey, UK). Thus, the substrate preference foreach antibody to different amyloid beta peptide preparations waspresented as the intrinsic affinity values (K_(D)).

Using this assay format, the interaction of the antibody and substratewas in the solution phase, thus, there was no constraint from any solidsurface. Further, the interactions were allowed to reach equilibrium.Therefore, the interaction of anti-ADDL antibody and substrate occurredat limiting concentrations of both components with no concerns forprecipitation of anti-ADDL antibody or additional amyloid beta peptideoligomerization due to high experimental concentration. Moreover, theassay readout was independent of antigen in the solution; thus, anyheterology of amyloid beta in different peptide preparations (e.g., ADDLor fibril) would not interfere with data interpretation and mathematicalmodeling. The assay sensitivity was limited to ELISA assay detectionlimits, which allowed this assay to evaluate monoclonal antibodies withK_(D) values in the nanomolar range.

The quantities of free anti-ADDL antibody were determined by a standardcurve and plotted against titrations of different substrates. Thequantities of bound anti-ADDL antibody with different substrates wereplotted and the information was used in GRAFIT for curve fitting withappropriate mathematic models. The summary of K_(D), expressed in nMranges, for the panel of anti-ADDL monoclonal antibodies is presented inTable 17.

TABLE 17 ADDL Fibril Aβ1-40 Aβ1-20 Antibody* K_(D) SE K_(D) SE K_(D) SEK_(D) SE 20C2 0.92 0.09 3.62 0.47 30.48 5.05 71.35 24.41 2A10 2.29 0.256.72 0.99 14.69 2.64 22.40 2.43 2B4 2.09 0.24 10.50 1.26 27.57 4.88 1.630.26 2D6 5.05 0.52 14.41 2.40 25.66 5.84 30.17 7.07 5F10 11.90 1.6328.95 5.78 23.54 6.21 6.10 4.39 4E2 4.26 0.42 9.40 1.60 20.24 2.07 28.403.23 4C2 8.08 1.03 19.17 3.69 21.89 4.14 28.40 3.23 1F4 9.24 0.84 12.521.66 IC IC IC IC 1F6 N/T N/T N/T N/T N/T N/T N/T N/T 2E12 IC IC IC IC ICIC IC IC WO-2 0.57 0.042 1.15 0.12 6.15 0.62 19.26 3.53 *All antibodieswere IgG. Values listed in italic are high SE and poor fitting. IC:inconclusive data. N/T: not tested.

What is claimed is:
 1. A kit for detecting oligomers of amyloid betacomprising: (a) a capture antibody that (i) recognizes an N-terminallinear epitope of amyloid beta 1-42 peptide, (ii) recognizes aconformational epitope of amyloid beta 1-42 oligomers, (iii) has ahigher affinity for amyloid beta 1-42 oligomers than for amyloid beta1-42 monomer, amyloid beta 1-40 monomer, plaques and amyloid betafibrils, (iv) exhibits less than a 10-fold decrease in EC₅₀ when storedat 40° C. for 1 month; and (b) a detection antibody that recognizes anN-terminal linear epitope of amyloid beta 1-42 peptide.
 2. The kit ofclaim 1, wherein the linear epitope of amyloid beta 1-42 for the captureantibody is within residues 1-20 of amyloid beta 1-42 or wherein thelinear epitope of amyloid beta 1-42 for the detection antibody is withinresidues 1-20 of amyloid beta 1-42.
 3. The kit of claim 1, wherein theaffinity of the capture antibody for amyloid beta 1-42 oligomerscompared to amyloid beta 1-40 monomers in a competitive binding assay isat least 500:1.
 4. The kit of claim 1, wherein the affinity of thecapture antibody for amyloid beta 1-42 oligomers compared to amyloidbeta 1-42 monomers in a sandwich ELISA assay is at least 500:1.
 5. Thekit of claim 1, wherein the affinity of the capture antibody for amyloidbeta 1-42 oligomers compared to amyloid beta 1-42 monomers in a sandwichELISA assay is at least 1000:1.
 6. The kit of claim 1, wherein the levelof detection of amyloid beta 1-42 oligomers by said kit is less than 5pg/mL.
 7. The kit of claim 1, wherein the level of detection of amyloidbeta 1-42 oligomers by said kit is less than 3 pg/mL.
 8. The kit ofclaim 1, wherein the capture antibody has (a) a light chain variableregion comprising, (i) a COR1 having the sequenceArg-Ser-Ser-Gln-Ser-Ile-Val-His-Ser-Xaa₁-Gly-Xaa₂-Thr-Thy-Leu-Glu (SEQID NO:1), wherein Xaa₁ is Asn, Ser, Thr, Ala, Asp or Glu and Xaa₂ isAsn, His, Gln, Ser, Thr, Ala, or Asp, (ii) a CDR2 having the sequenceLys-Ala-Ser-Xaa₁-Arg-Phe-Ser (SEQ ID NO:2), wherein Xaa₁ is Asn, Gly,Ser, Thr, or Ala, and (iii) a CDR3 having the sequencePhe-Gln-Gly-Ser-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅ (SEQ ID NO:3), wherein Xaa₁ isArg, Lys or Tyr, Xaa₂ is Val, Ala, or Leu, Xaa₃ is Pro, His, or Gly,Xaa₄ is Ala, Pro, or Val, and Xaa₅ is Ser, Gly, or Phe; and (b) a heavychain variable region comprising, (i) a CDR1 of SEQ ID NO:4, (ii) a CDR2of SEQ ID NO:5, and (iii) a CDR3 of SEQ ID NO:6.
 9. The kit of claim 8,wherein the CDR1 of the light chain variable region has the sequenceArg-Ser-Ser-Gln-Ser-Ile-Val-His-Ser-Xaa₁-Gly-Xaa₂-Thr-Thy-Leu-Glu (SEQID NO:1), wherein Xaa₁ is Thr, Ala, Asp or Glu and Xaa₂ is Thr.
 10. Thekit of claim 8, wherein the CDR2 of the light chain variable region hasthe sequence Lys-Ala-Ser-Xaa₁-Arg-Phe-Ser (SEQ ID NO:2), wherein Xaa₁ isThr.
 11. The kit of claim 1, wherein the detection antibody is 6E10,BAM-10, W0-2, 26D6, 2A10, 2B4, 4C2, 4E2, 2H4, 20C2, 2D6, 5F10, 1F4, 1F6,2E12, 3B3 or 82E1.
 12. The kit of claim 1, wherein the detectionantibody further comprises a label.
 13. The kit of claim 1, furthercomprising a means for concentrating an antibody-antigen complex.
 14. Amethod for detecting oligomers of amyloid beta comprising: (a)contacting a biological sample from an animal, said biological samplehaving oligomers of amyloid beta, with a capture antibody underconditions sufficient to form a capture antibody/oligomer of amyloidbeta complex, wherein the capture antibody (i) recognizes an N-terminallinear epitope of amyloid beta 1-42 peptide, (ii) recognizes aconformational epitope of amyloid beta 1-42 oligomers, (iii) has ahigher affinity for amyloid beta 1-42 oligomers than for amyloid beta1-42 monomer, amyloid beta 1-40 monomer, plaques and amyloid betafibrils, (iv) exhibits less than a 10-fold decrease in EC₅₀ when storedat 40° C. for 1 month; (b) contacting the complex of step (a) with adetection antibody under conditions sufficient to form captureantibody/oligomer of amyloid beta/detection antibody complex, whereinthe detection antibody recognizes an N-terminal linear epitope ofamyloid beta 1-42 peptide; and (c) detecting the complex of step (b).15. A method of claim 14, wherein the animal is a human.
 16. The methodof claim 14, wherein the linear epitope of amyloid beta 1-42 for thecapture antibody is within residues 1-20 of amyloid beta 1-42 or whereinthe linear epitope of amyloid beta 1-42 for the detection antibody iswithin residues 1-20 of amyloid beta 1-42.
 17. The method of claim 14,wherein the affinity of the capture antibody for amyloid beta 1-42oligomers compared to amyloid beta 1-40 monomers in a competitivebinding assay is at least 500:1.
 18. The method of claim 14, wherein theaffinity of the capture antibody for amyloid beta 1-42 oligomerscompared to amyloid beta 1-42 monomers in a sandwich ELISA assay is atleast 500:1.
 19. The method of claim 14, wherein the affinity of thecapture antibody for amyloid beta 1-42 oligomers compared to amyloidbeta 1-42 monomers in a sandwich ELISA assay is at least 1000:1.
 20. Themethod of claim 14, wherein the biological sample comprises cerebralspinal fluid.
 21. The method of claim 20, wherein said method is capableof detecting less than 5 pg/mL of amyloid beta 1-42 oligomers in thecerebral spinal fluid.
 22. The method of claim 20, wherein said methodis capable of detecting less than 3 pg/mL of amyloid beta 1-42 oligomersin the cerebral spinal fluid.
 23. The method of claim 14, wherein thecapture antibody has (a) a light chain variable region comprising, (i) aCDR1 having the sequenceArg-Ser-Ser-Gln-Ser-Ile-Val-His-Ser-Xaa₁-Gly-Xaa₂-Thr-Thy-Leu-Glu (SEQID NO:1), wherein Xaa₁ is Asn, Ser, Thr, Ala, Asp or Glu and Xaa₂ isAsn, His, Gln, Ser, Thr, Ala, or Asp, (ii) a CDR2 having the sequenceLys-Ala-Ser-Xaa₁-Arg-Phe-Ser (SEQ ID NO:2), wherein Xaa₁ is Asn, Gly,Ser, Thr, or Ala, and (iii) a CDR3 having the sequencePhe-Gln-Gly-Ser-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅ (SEQ ID NO:3), wherein Xaa₁ isArg, Lys or Tyr, Xaa₂ is Val, Ala, or Leu, Xaa₃ is Pro, His, or Gly,Xaa₄ is Ala, Pro, or Val, and Xaa₅ is Ser, Gly, or Phe; and (b) a heavychain variable region comprising, (i) a CDR1 of SEQ ID NO:4, (ii) a CDR2of SEQ ID NO:5, and (iii) a CDR3 of SEQ ID NO:6.
 24. The method of claim23, wherein the CDR1 of the light chain variable region has the sequenceArg-Ser-Ser-Gln-Ser-Ile-Val-His-Ser-Xaa₁-Gly-Xaa₂-Thr-Thy-Leu-Glu (SEQID NO:1), wherein Xaa₁ is Thr, Ala, Asp or Glu and Xaa₂ is Thr.
 25. Themethod of claim 23, wherein the CDR2 of the light chain variable regionhas the sequence Lys-Ala-Ser-Xaa₁-Arg-Phe-Ser (SEQ ID NO:2), whereinXaa₁ is Thr.
 26. The method of claim 14, wherein the detection antibodyis 6E10, BAM-10, W0-2, 26D6, 2A10, 2B4, 4C2, 4E2, 2H4, 20C2, 2D6, 5F10,1F4, 1F6, 2E12, 3B3 or 82E1.
 27. The method of claim 14, wherein thedetection antibody further comprises a label.
 28. The method of claim14, further comprising the step of concentrating the captureantibody/oligomer of amyloid beta/detection antibody complex prior todetecting the complex.
 29. An isolated antibody, or antigen bindingfragment thereof, that binds amyloid β-derived diffusible ligandscomprising: (a) a light chain variable region comprising, (i) a CDR1having the sequenceArg-Ser-Ser-Gln-Ser-Ile-Val-His-Ser-Xaa₁-Gly-Xaa₂-Thr-Thy-Leu-Glu (SEQID NO:1), wherein Xaa₁ is Asn, Ser, Thr, Ala, Asp or Glu and Xaa₂ isAsn, His, Gln, Ser, Thr, Ala, or Asp, (ii) a CDR2 of SEQ ID NO:15, and(iii) a CDR3 of SEQ ID NO:16; and (b) a heavy chain variable regioncomprising, (i) a CDR1 of SEQ ID NO:4, (ii) a CDR2 of SEQ ID NO:5, and(iii) a CDR3 of SEQ ID NO:6.
 30. The antibody of claim 29, wherein theCDR1 of the light chain variable region has the sequenceArg-Ser-Ser-Gln-Ser-Ile-Val-His-Ser-Xaa₁-Gly-Xaa₂-Thr-Thy-Leu-Glu (SEQID NO:1), wherein Xaa₁ is Thr, Ala, Asp or Glu and Xaa₂ is Thr.
 31. Anisolated antibody, or antigen binding fragment thereof, that bindsamyloid β-derived diffusible ligands comprising: (a) a light chainvariable region comprising, (i) a CDR1 of SEQ ID NO:14, (ii) a CDR2having the sequence Lys-Ala-Ser-Xaa₁-Arg-Phe-Ser (SEQ ID NO:2), whereinXaa₁ is Asn, Gly, Ser, Thr, or Ala, and (iii) a CDR3 of SEQ ID NO:16;and (b) a heavy chain variable region comprising, (i) a CDR1 of SEQ IDNO:4, (ii) a CDR2 of SEQ ID NO:5, and (iii) a CDR3 of SEQ ID NO:6. 32.The antibody of claim 31, wherein the CDR2 of the light chain variableregion has the sequence Lys-Ala-Ser-Xaa₁-Arg-Phe-Ser (SEQ ID NO:2),wherein Xaa₁ is Thr.